Fuel compositions comprising isoprene derivatives

ABSTRACT

The invention provides for methods, compositions and systems using bioisoprene derived from renewable carbon for production of a variety of hydrocarbon fuels and fuel additives.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 61/187,959, filed Jun. 17, 2009, the disclosure of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The development of renewable transportation fuels is one of the key challenges of the twenty-first century. The current market is dominated by ethanol derived from yeast fermentation of sucrose and starch, and to a lesser extent by biodiesel (fatty acid esters) derived from triglycerides. Ethanol has limitations as a liquid fuel with a lower energy density relative to hydrocarbons. In addition, ethanol cannot be transported in conventional infrastructure due to its affinity for water and corrosive nature. Processes for the conversion of renewable carbon sources (biomass, sugars, oils) to hydrocarbon fuels offer an attractive alternative to bioethanol.

Isoprene (2-methyl-1,3-butadiene) is a key industrial chemical used primarily for the production of synthetic rubber. Currently isoprene is derived from petrochemical sources either directly by cracking of naphtha and other light petroleum fractions, or indirectly through chemical synthesis (See, for examples, H. Pommer and A. Nurrenbach, Industrial Synthesis of Terpene Compounds, Pure Appl. Chem., 1975, 43, 527-551; H. M. Weitz and E. Loser, Isoprene, in Ullmann's Encyclopedia of Industrial Chemistry, Seventh Edition, Electronic Release, Wiley-VCH Verlag GMBH, Weinheim, 2005; and H. M. Lybarger, Isoprene in Kirk-Othmer Encyclopedia of Chemical Technology, 4th ed., Wiley, New York (1995), 14, 934-952.) The resulting crude isoprene streams are typically subjected to extensive purification processes in order to remove numerous chemically similar impurities, many of which can interfere with subsequent transformation of isoprene to polymers and other chemicals.

In contrast, isoprene derived from biological sources contains very few hydrocarbon impurities and instead contains a number of oxygenated compounds such as ethanol, acetaldehyde and acetone. Many of these compounds can be easily removed by contact with water or passage through alumina or other adsorbents.

Industry relies on petrochemical feedstocks for isoprene production and extensive purification trains are needed before isoprene can be converted to polymers and other chemicals. Cost effective methods are desirable for converting biologically produced isoprene to valuable chemical products taking advantage of the high purity and/or the unique impurity profiles of bioisoprene.

All patents, patent applications, documents, and articles cited herein are herein incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

Disclosed are methods and systems for producing fuel constituents from highly pure isoprene and fuel compositions produced from highly pure isoprene.

In one aspect, the invention provides a method for producing a fuel constituent from a bioisoprene composition comprising chemically transforming a substantial portion of the isoprene in the bioisoprene composition to non-isoprene compounds. In one embodiment, the bioisoprene composition is chemically transformed by subjecting the bioisoprene composition to heat or catalytic conditions suitable for isoprene dimerization to produce an isoprene dimer and then catalytically hydrogenating the isoprene dimer to form a saturated C10 fuel constituent. In another embodiment, the bioisoprene composition is chemically transformed by (i) partially hydrogenating the bioisoprene composition to produce an isoamylene, (ii) dimerizing the isoamylene with a mono-olefin selected from the group consisting of isoamylene, propylene and isobutene to form a dimate and (iii) completely hydrogenating the dimate to produce a fuel constituent. In some embodiments, at least about 95% of isoprene in the bioisoprene composition is converted to non-isoprene compounds during the chemical transformation. In some embodiments, the bioisoprene composition is heated to about 150° C. to about 250° C. to produce an unsaturated cyclic isoprene dimer and the unsaturated cyclic isoprene dimer is hydrogenated catalytically to produce a saturated cyclic isoprene dimer fuel constituent. In some embodiments, the method comprises: (i) contacting the bioisoprene composition with a catalyst for catalyzing cyclo-dimerization of isoprene to produce an unsaturated cyclic isoprene dimer and the unsaturated cyclic isoprene dimer is hydrogenated catalytically to produce a saturated cyclic isoprene dimer fuel constituent. In some embodiments, the catalyst for catalyzing cyclo-dimerization of isoprene comprising a catalyst selected from the group consisting of a nickel catalyst, iron catalysts and chromium catalysts. In some embodiments, the step of partially hydrogenating the bioisoprene composition comprises contacting the bioisoprene composition with hydrogen gas and a catalyst for catalyzing partial hydrogenation of isoprene. In some embodiments, the catalyst for catalyzing partial hydrogenation of isoprene comprises a palladium catalyst. In some embodiments, the step of dimerizing the isoamylene with a mono-olefin comprises contacting the isoamylene with the mono-olefin in the presence of a catalyst for catalyzing dimerization of mono-olefin. In some embodiments, the catalyst for catalyzing dimerization of mono-olefin comprises an acid catalyst. In some embodiments, the method further comprises purifying the isoprene from the bioisoprene composition prior to chemically transforming the bioisoprene composition to a fuel constituent.

In one aspect, the invention provides a system for producing a fuel constituent from a bioisoprene composition, wherein a substantial portion of the isoprene in the bioisoprene composition is chemically converted to non-isoprene compounds, the system comprising a bioisoprene composition and (a) (i) one or more chemicals capable of dimerizing isoprene in the bioisoprene composition or a source of heat capable of dimerizing isoprene in the bioisoprene composition; and (ii) a catalyst capable of hydrogenating the isoprene dimer to form a saturated C10 fuel constituent; or (b) (i) a chemical capable of partially hydrogenating isoprene in the bioisoprene composition to produce an isoamylene, (ii) a chemical capable of dimerizing the isoamylene with mono-olefins selected from the group consisting of isoamylene, propylene and isobutene to form a dimate and (iii) a chemical capable of completely hydrogenating the dimate to produce a fuel constituent.

In some embodiments of the system, the bioisoprene composition comprising greater than about 2 mg of isoprene and comprising greater than or about 99.94% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments of the system, the one or more chemicals capable of dimerizing isoprene comprises catalyst for catalyzing cyclo-dimerization of isoprene comprising a catalyst selected from the group consisting of ruthenium catalysts, nickel catalysts, iron catalysts and chromium catalysts. In some embodiments of the system, the catalyst for hydrogenating the unsaturated isoprene dimers comprises a catalyst selected from the group consisting of palladium catalysts, nickel catalysts, ruthenium catalysts and rhodium catalysts. In some embodiments of the system, the chemical capable of partially hydrogenating isoprene comprises a palladium catalyst. In some embodiments of the system, the chemical capable of dimerizing the isoamylene with mono-olefins comprises an acid catalyst.

In one aspect, the invention provides a fuel composition comprising a fuel constituent produced by the methods described herein. In some embodiments, the fuel composition is substantially free of isoprene. In some embodiments, the fuel composition has δ¹³C value which is greater than −22‰ or within the range of −32‰ to −24‰.

In some aspects, the invention provides a system for producing a fuel constituent from isoprene comprising: (a) a commercially beneficial amount of highly pure isoprene; and (b) a fuel constituent produced from at least a portion of the highly pure isoprene; wherein at least a portion of the commercially beneficial amount of highly pure isoprene undergoes a chemical transformation.

In some embodiments of the system, the commercially beneficial amount of highly pure isoprene comprises greater than about 2 mg of isoprene and comprising greater than or about 99.94% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the commercially beneficial amount of highly pure isoprene comprises greater than about 2 mg of isoprene and comprising one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms. In some embodiments, the commercially beneficial amount of highly pure isoprene comprises greater than about 2 mg of isoprene and comprising one or more second compounds selected from the group consisting of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol, 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine; wherein the amount of the second compound relative to the amount of the isoprene is greater than or about 0.01% (w/w). In some embodiments, the commercially beneficial amount of highly pure isoprene comprises greater than about 2 mg of isoprene and comprising less than or about 0.5 μg/L per compound for any compound in the composition that inhibits the polymerization of isoprene. In some preferred embodiments, the commercially beneficial amount of highly pure isoprene is produced by a biological process.

In some embodiments of the system, the fuel constituent comprises one or more compounds selected from the group consisting of cyclic isoprene dimers and trimers, linear isoprene oligomers, aromatic and alicyclic isoprene derivatives, and oxygenated isoprene derivatives. In some embodiments, the oxygenated isoprene derivatives are compounds selected from the group consisting of alcohols, ketones, esters and ethers derived from isoprene.

In some embodiments, the fuel constituent comprises cyclic isoprene dimers and the chemical transformation comprises a dimerization reaction of isoprene. In some embodiments, the dimerization reaction is carried out by heating the commercially beneficial amount of highly pure isoprene. In some embodiments, the dimerization reaction of isoprene produces a product comprising unsaturated isoprene dimers and the chemical transformation further comprises a hydrogenation reaction of the unsaturated isoprene dimers. In some embodiments, the system further comprises a catalyst for catalyzing the hydrogenation reaction of the unsaturated isoprene dimers. In some embodiments, the catalyst for catalyzing the hydrogenation reaction of the unsaturated isoprene dimers comprising a catalyst selected from the group consisting of palladium catalysts, nickel catalysts, ruthenium catalysts and rhodium catalysts.

In some embodiments, the dimerization reaction is carried out by contacting the commercially beneficial amount of highly pure isoprene with a catalyst for catalyzing cyclo-dimerization of isoprene. In some embodiments, the catalyst for catalyzing cyclo-dimerization of isoprene comprising a catalyst selected from the group consisting of ruthenium catalysts, nickel catalysts, iron catalysts and chromium catalysts. In some embodiments, the catalyst for catalyzing cyclo-dimerization of isoprene is a nickel catalyst and the fuel constituent comprising one or more eight-membered ring dimers of isoprene.

In some embodiments, the fuel constituent comprises linear and/or cyclic trimers of isoprene and the chemical transformation comprises catalytic trimerization of isoprene.

In one aspect, the invention provides a method for producing a fuel constituent from isoprene comprising: (a) obtaining a commercially beneficial amount of highly pure isoprene; and (b) chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent. In some embodiments, the commercially beneficial amount of highly pure isoprene comprises bioisoprene.

In some embodiments, the commercially beneficial amount of highly pure isoprene is obtained by the steps comprising: (i) culturing cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide under suitable culture conditions for the production of isoprene, wherein the cells (1) produce greater than about 400 nmole/g_(wcm)/hr of isoprene, (2) convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, or (3) have an average volumetric productivity of isoprene greater than about 0.1 mg/L_(broth)/hr of isoprene, and (ii) producing isoprene. In some embodiments, the cells further comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide or an MVA pathway polypeptide.

In some embodiments of the methods described herein, chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent comprises: (i) heating the commercially beneficial amount of highly pure isoprene to about 150° C. to about 250° C.; (ii) converting at least a portion of the commercially beneficial amount of highly pure isoprene to unsaturated cyclic isoprene dimers; (iii) hydrogenating the unsaturated cyclic isoprene dimers to produce saturated cyclic isoprene dimers; and (iv) producing the fuel constituent. In some embodiments, at least about 20% to about 100% of isoprene in the commercially beneficial amount of highly pure isoprene is converted to unsaturated cyclic isoprene dimers.

In some embodiments of the method, chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent comprises: (i) contacting the commercially beneficial amount of highly pure isoprene with a catalyst for catalyzing cyclo-dimerization of isoprene, (ii) converting at least a portion of the commercially beneficial amount of highly pure isoprene to cyclic isoprene dimers; and (iii) producing the fuel constituent.

In some embodiments of the method, chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent comprises: (i) contacting the commercially beneficial amount of highly pure isoprene with a catalyst for catalyzing cyclo-dimerization of isoprene, (ii) converting at least a portion of the commercially beneficial amount of highly pure isoprene to unsaturated cyclic isoprene dimers; (iii) hydrogenating the unsaturated cyclic isoprene dimers to produce saturated cyclic isoprene dimers; and (iv) producing the fuel constituent. In some embodiments of the method, the catalyst for catalyzing cyclo-dimerization of isoprene comprising a catalyst selected from the group consisting of a nickel catalyst, iron catalysts and chromium catalysts.

In some embodiments of the method, chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent comprises: (i) contacting the highly pure isoprene composition with a catalyst system; (ii) converting at least a portion of the starting isoprene composition to unsaturated isoprene dimers and/or trimers; and (iii) hydrogenating the unsaturated dimers and/or trimers to produce saturated C10 and/or C15 hydrocarbons.

In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) converting at least a portion of the starting isoprene composition to oxygenated isoprene derivatives; and optionally (c) hydrogenating any unsaturated oxygenated isoprene derivatives to produce saturated oxygenates. In some embodiments, the oxygenated isoprene derivatives are compounds selected from the group consisting of alcohols, ketones, esters and ethers derived from isoprene.

In some embodiments, any of the methods described herein further comprises purifying the commercially beneficial amount of highly pure isoprene prior to chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent.

In one aspect, provided is a continuous process for producing a fuel constituent from isoprene comprising: (a) continuously producing a commercially beneficial amount of highly pure isoprene; and (b) continuously transforming chemically at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent. In some embodiments, the commercially beneficial amount of highly pure isoprene comprising a gas phase comprising isoprene. In some embodiments, the method further comprises passing the gas phase comprising isoprene to a reactor for chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent. In a preferred embodiment, the commercially beneficial amount of highly pure isoprene comprises bioisoprene.

Also provided is a fuel composition comprising a fuel constituent produced by any of the methods described herein. In some embodiments, the fuel constituent comprises less than or about 0.5 μg/L a product from a C5 hydrocarbon other than isoprene after undergoing the steps according to the methods described herein. In some embodiments, the fuel constituent comprises one or more product from one or more compound selected from the group consisting of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol, 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene and 2,3-cycloheptenolpyridine after undergoing the steps according to the methods described herein.

In some embodiments, the fuel composition comprises a fuel composition having δ¹³C value which is greater than −22‰. In some embodiments, the fuel composition has δ¹³C value which is within the range of −22‰ to −10‰ or −34‰ to −24‰. In some embodiments, the fuel composition has f_(M) value which is greater than 0.9. Also provided is a blend of any of the fuel compositions described herein with a petroleum based fuel in the amount of from about 1% to about 95% by weight or volume, based on the total weight or volume of the total fuel composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleotide sequence of a kudzu isoprene synthase gene codon-optimized for expression in E. coli (SEQ ID NO:1). The atg start codon is in italics, the stop codon is in bold and the added PstI site is underlined.

FIG. 2 is a map of pTrcKudzu.

FIGS. 3A-C are the nucleotide sequence of pTrcKudzu (SEQ ID NO:2). The RBS is underlined, the kudzu isoprene synthase start codon is in bold capitol letters and the stop codon is in bold, capital letters. The vector backbone is pTrcHis2B.

FIG. 4 is a map of pETNHisKudzu.

FIGS. 5A-C are the nucleotide sequence of pETNHisKudzu (SEQ ID NO:3).

FIG. 6 is a map of pCL-lac-Kudzu.

FIGS. 7A-C are the nucleotide sequence of pCL-lac-Kudzu (SEQ ID NO:4).

FIG. 8A is a graph showing the production of isoprene in E. coli BL21 cells with no vector.

FIG. 8B is a graph showing the production of isoprene in E. coli BL21 cells with pCL-lac-Kudzu

FIG. 8C is a graph showing the production of isoprene in E. coli BL21 cells with pTrcKudzu.

FIG. 8D is a graph showing the production of isoprene in E. coli BL21 cells with pETN-HisKudzu.

FIG. 9A is a graph showing OD over time of fermentation of E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 9B is a graph showing isoprene production over time of fermentation of E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 10A is a graph showing the production of isoprene in Panteoa citrea. Control cells without recombinant kudzu isoprene synthase. Grey diamonds represent isoprene synthesis, black squares represent OD₆₀₀.

FIG. 10B is a graph showing the production of isoprene in Panteoa citrea expressing pCL-lac Kudzu. Grey diamonds represent isoprene synthesis, black squares represent OD₆₀₀.

FIG. 10C is a graph showing the production of isoprene in Panteoa citrea expressing pTrcKudzu. Grey diamonds represent isoprene synthesis, black squares represent OD₆₀₀.

FIG. 11 is a graph showing the production of isoprene in Bacillus subtilis expressing recombinant isoprene synthase. BG3594comK is a B. subtilis strain without plasmid (native isoprene production). CF443 is B. subtilis strain BG3594comK with pBSKudzu (recombinant isoprene production). IS on the y-axis indicates isoprene.

FIGS. 12A-C are the nucleotide sequence of pBS Kudzu #2 (SEQ ID NO:5).

FIG. 13 is the nucleotide sequence of kudzu isoprene synthase codon-optimized for expression in Yarrowia (SEQ ID NO:6).

FIG. 14 is a map of pTrex3g comprising a kudzu isoprene synthase gene codon-optimized for expression in Yarrowia.

FIGS. 15A-C are the nucleotide sequence of vector pSPZ1(MAP29Spb) (SEQ ID NO:7).

FIG. 16 is the nucleotide sequence of the synthetic kudzu (Pueraria montana) isoprene gene codon-optimized for expression in Yarrowia (SEQ ID NO:8).

FIG. 17 is the nucleotide sequence of the synthetic hybrid poplar (Populus alba x Populus tremula) isoprene synthase gene (SEQ ID NO:9). The ATG start codon is in bold and the stop codon is underlined.

FIG. 18A (FIGS. 18A1 and 18A2) shows a schematic outlining construction of vectors pYLA 1, pYL1 and pYL2 (SEQ ID NO:75, 73, 72, 71, 70, 69).

FIG. 18B shows a schematic outlining construction of the vector pYLA(POP1) (SEQ ID NO: 67, 68).

FIG. 18C shows a schematic outlining construction of the vector pYLA(KZ1)

FIG. 18D shows a schematic outlining construction of the vector pYLI(KZ1) (SEQ ID NO: 65, 66).

FIG. 18E shows a schematic outlining construction of the vector pYLI(MAP29)

FIG. 18F shows a schematic outlining construction of the vector pYLA(MAP29)

FIG. 19A shows the MVA and DXP metabolic pathways for isoprene (based on F. Bouvier et al., Progress in Lipid Res. 44: 357-429, 2005). The following description includes alternative names for each polypeptide in the pathways and a reference that discloses an assay for measuring the activity of the indicated polypeptide (each of these references are each hereby incorporated by reference in their entireties, particularly with respect to assays for polypeptide activity for polypeptides in the MVA and DXP pathways). Mevalonate Pathway: AACT; Acetyl-CoA acetyltransferase, MvaE, EC 2.3.1.9. Assay: J. Bacteriol., 184: 2116-2122, 2002; HMGS; Hydroxymethylglutaryl-CoA synthase, MvaS, EC 2.3.3.10. Assay: J. Bacteriol., 184: 4065-4070, 2002; HMGR; 3-Hydroxy-3-methylglutaryl-CoA reductase, MvaE, EC 1.1.1.34. Assay: J. Bacteriol., 184: 2116-2122, 2002; MVK; Mevalonate kinase, ERG12, EC 2.7.1.36. Assay: Curr Genet. 19:9-14, 1991. PMK; Phosphomevalonate kinase, ERGS, EC 2.7.4.2, Assay: Mol Cell Biol., 11:620-631, 1991; DPMDC; Diphosphomevalonate decarboxylase, MVD1, EC 4.1.1.33. Assay: Biochemistry, 33:13355-13362, 1994; IDI; Isopentenyl-diphosphate delta-isomerase, IDI1, EC 5.3.3.2. Assay: J. Biol. Chem. 264:19169-19175, 1989. DXP Pathway: DXS; 1-Deoxyxylulose-5-phosphate synthase, dxs, EC 2.2.1.7. Assay: PNAS, 94:12857-62, 1997; DXR; 1-Deoxy-D-xylulose 5-phosphate reductoisomerase, dxr, EC 2.2.1.7. Assay: Eur. J. Biochem. 269:4446-4457, 2002; MCT; 4-Diphosphocytidyl-2C-methyl-D-erythritol synthase, IspD, EC 2.7.7.60. Assay: PNAS, 97: 6451-6456, 2000; CMK; 4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase, IspE, EC 2.7.1.148. Assay: PNAS, 97:1062-1067, 2000; MCS; 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase, IspF, EC 4.6.1.12. Assay: PNAS, 96:11758-11763, 1999; HDS; 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, ispG, EC 1.17.4.3. Assay: J. Org. Chem., 70:9168-9174, 2005; HDR; 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, IspH, EC 1.17.1.2. Assay: JACS, 126:12847-12855, 2004.

FIG. 19B illustrates the classical and modified MVA pathways. 1, acetyl-CoA acetyltransferase (AACT); 2, HMG-CoA synthase (HMGS); 3, HMG-CoA reductase (HMGR); 4, mevalonate kinase (MVK); 5, phosphomevalonate kinase (PMK); 6, diphosphomevalonate decarboxylase (MVD or DPMDC); 7, isopentenyl diphosphate isomerase (IDI); 8, phosphomevalonate decarboxylase (PMDC); 9, isopentenyl phosphate kinase (IPK). The classical MVA pathway proceeds from reaction 1 through reaction 7 via reactions 5 and 6, while a modified MVA pathway goes through reactions 8 and 9. P and PP in the structural formula are phosphate and pyrophosphate, respectively. This figure was taken from Koga and Morii, Microbiology and Mol. Biology. Reviews, 71:97-120, 2007, which is incorporated by reference in its entirety, particular with respect to nucleic acids and polypeptides of the modified MVA pathway. The modified MVA pathway is present, for example, in some archaeal organisms, such as Methanosarcina mazei.

FIG. 20 (FIGS. 20A and 20B) shows graphs representing results of the GC-MS analysis of isoprene production by recombinant Y. lipolytica strains without (left) or with (right) a kudzu isoprene synthase gene. The arrows indicate the elution time of the authentic isoprene standard.

FIG. 21 is a map of pTrcKudzu yIDI DXS Kan.

FIGS. 22A-D are the nucleotide sequence of pTrcKudzu yIDI DXS Kan (SEQ ID NO:10).

FIG. 23A is a graph showing production of isoprene from glucose in BL21/pTrcKudzukan. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).

FIG. 23B is a graph showing production of isoprene from glucose in BL21/pTrcKudzu yIDI kan. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).

FIG. 23C is a graph showing production of isoprene from glucose in BL21/pTrcKudzu DXS kan. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).

FIG. 23D is a graph showing production of isoprene from glucose in BL21/pTrcKudzu yIDI DXS kan. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).

FIG. 23E is a graph showing production of isoprene from glucose in BL21/pCL PtrcKudzu. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).

FIG. 23F is a graph showing production of isoprene from glucose in BL21/pCL PtrcKudzu yIDI. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).

FIG. 23G is a graph showing production of isoprene from glucose in BL21/pCL PtrcKudzu DXS. Time 0 is the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circles represent total isoprene productivity (μg/L) and squares represent specific productivity of isoprene (μg/L/OD).

FIG. 23H is a graph showing production of isoprene from glucose in BL21/pTrcKudzuIDIDXSkan. The arrow indicates the time of induction with IPTG (400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axis is total productivity of isoprene (μg/L headspace or specific productivity (μg/L headspace/OD). Black diamonds represent OD₆₀₀, black triangles represent isoprene productivity (μg/L) and white squares represent specific productivity of isoprene (μg/L/OD).

FIG. 24 is a map of pTrcKKDyIkIS kan.

FIGS. 25A-D are a nucleotide sequence of pTrcKKDyIkIS kan (SEQ ID NO:11).

FIG. 26 is a map of pCL PtrcUpperPathway.

FIGS. 27A-27D is a nucleotide sequence of pCL PtrcUpperPathway (SEQ ID NO:12).

FIG. 28 shows a map of the cassette containing the lower MVA pathway and yeast idi for integration into the B. subtilis chromosome at the nprE locus. nprE upstream/downstream indicates 1 kb each of sequence from the nprE locus for integration. aprE promoter (alkaline serine protease promoter) indicates the promoter (−35, −10, +1 transcription start site, RBS) of the aprE gene. MVK1 indicates the yeast mevalonate kinase gene. RBS-PMK indicates the yeast phosphomevalonate kinase gene with a Bacillus RBS upstream of the start site. RBS-MPD indicates the yeast diphosphomevalonate decarboxylase gene with a Bacillus RBS upstream of the start site. RBS-IDI indicates the yeast idi gene with a Bacillus RBS upstream of the start site. Terminator indicates the terminator alkaline serine protease transcription terminator from B. amyliquefaciens. SpecR indicates the spectinomycin resistance marker. “nprE upstream repeat for amp.” indicates a direct repeat of the upstream region used for amplification.

FIGS. 29A-D are a nucleotide sequence of cassette containing the lower MVA pathway and yeast idi for integration into the B. subtilis chromosome at the nprE locus (SEQ ID NO:13).

FIG. 30 is a map of p9796-poplar.

FIGS. 31A-B are a nucleotide sequence of p9796-poplar (SEQ ID NO:14).

FIG. 32 is a map of pTrcPoplar.

FIGS. 33A-C are a nucleotide sequence of pTrcPoplar (SEQ ID NO:15).

FIG. 34 is a map of pTrcKudzu yIDI Kan.

FIGS. 35A-C are a nucleotide sequence of pTrcKudzu yIDI Kan (SEQ ID NO:16).

FIG. 36 is a map of pTrcKudzuDXS Kan.

FIGS. 37A-C are a nucleotide sequence of pTrcKudzuDXS Kan (SEQ ID NO:17).

FIG. 38 is a map of pCL PtrcKudzu.

FIGS. 39A-C are a nucleotide sequence of pCL PtrcKudzu (SEQ ID NO:18).

FIG. 40 is a map of pCL PtrcKudzu A3.

FIGS. 41A-C are a nucleotide sequence of pCL PtrcKudzu A3 (SEQ ID NO:19).

FIG. 42 is a map of pCL PtrcKudzu yIDI.

FIGS. 43A-C are a nucleotide sequence of pCL PtrcKudzu yIDI (SEQ ID NO:20).

FIG. 44 is a map of pCL PtrcKudzu DXS.

FIGS. 45A-D are a nucleotide sequence of pCL PtrcKudzu DXS (SEQ ID NO:21).

FIGS. 46A-E show graphs representing isoprene production from biomass feedstocks. Panel A shows isoprene production from corn stover, Panel B shows isoprene production from bagasse, Panel C shows isoprene production from softwood pulp, Panel D shows isoprene production from glucose, and Panel E shows isoprene production from cells with no additional feedstock. Grey squares represent OD₆₀₀ measurements of the cultures at the indicated times post-inoculation and black triangles represent isoprene production at the indicated times post-inoculation.

FIG. 47A shows a graph representing isoprene production by BL21 (λDE3) pTrcKudzu yIDI DXS (kan) in a culture with no glucose added. Squares represent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47B shows a graph representing isoprene production from 1% glucose feedstock invert sugar by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squares represent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47C shows a graph representing isoprene production from 1% invert sugar feedstock by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squares represent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47D shows a graph representing isoprene production from 1% AFEX corn stover feedstock by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squares represent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIGS. 48A-C show graphs demonstrating the effect of yeast extract of isoprene production. Panel A shows the time course of optical density within fermentors fed with varying amounts of yeast extract. Panel B shows the time course of isoprene titer within fermentors fed with varying amounts of yeast extract. The titer is defined as the amount of isoprene produced per liter of fermentation broth. Panel C shows the effect of yeast extract on isoprene production in E. coli grown in fed-batch culture.

FIGS. 49A-C show graphs demonstrating isoprene production from a 500 L bioreactor with E. coli cells containing the pTrcKudzu+yIDI+DXS plasmid. Panel A shows the time course of optical density within the 500-L bioreactor fed with glucose and yeast extract. Panel B shows the time course of isoprene titer within the 500-L bioreactor fed with glucose and yeast extract. The titer is defined as the amount of isoprene produced per liter of fermentation broth. Panel C shows the time course of total isoprene produced from the 500-L bioreactor fed with glucose and yeast extract.

FIG. 50 is a map of pJMupperpathway2.

FIGS. 51A-C are the nucleotide sequence of pJMupperpathway2 (SEQ ID NO:22).

FIG. 52 is a map of pBS Kudzu #2.

FIG. 53A is a graph showing growth during fermentation time of Bacillus expressing recombinant kudzu isoprene synthase in 14 liter fed batch fermentation. Black diamonds represent a control strain (BG3594comK) without recombinant isoprene synthase (native isoprene production) and grey triangles represent CF443, Bacillus strain BG3594comK with pBSKudzu (recombinant isoprene production).

FIG. 53B is a graph showing isoprene production during fermentation time of Bacillus expressing recombinant kudzu isoprene synthase in 14 liter fed batch fermentation. Black diamonds represent a control strain (BG3594comK) without recombinant isoprene synthase (native isoprene production) and grey triangles represent CF443, Bacillus strain BG3594comK with pBSKudzu (recombinant isoprene production).

FIG. 54 is a time course of optical density within the 15-L bioreactor fed with glucose.

FIG. 55 is a time course of isoprene titer within the 15-L bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

FIG. 56 is a time course of total isoprene produced from the 15-L bioreactor fed with glucose.

FIG. 57 is a time course of optical density within the 15-L bioreactor fed with glycerol.

FIG. 58 is a time course of isoprene titer within the 15-L bioreactor fed with glycerol. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

FIG. 59 is a time course of total isoprene produced from the 15-L bioreactor fed with glycerol.

FIGS. 60A-60C are the time courses of optical density, mevalonic acid titer, and specific productivity within the 150-L bioreactor fed with glucose.

FIGS. 61A-61C are the time courses of optical density, mevalonic acid titer, and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 62A-62C are the time courses of optical density, mevalonic acid titer, and specific productivity within the 15-L bioreactor fed with glucose.

FIG. 63A-63C are the time courses of optical density, isoprene titer, and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 64A-64C are the time courses of optical density, isoprene titer, and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 65A-65C are the time courses of optical density, isoprene titer, and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 66A-66C are the time courses of optical density, isoprene titer, and specific productivity within the 15-L bioreactor fed with glucose.

FIG. 67A-67C are the time courses of optical density, isoprene titer, and specific productivity within the 15-L bioreactor fed with glucose.

FIG. 68 is a graph of the calculated adiabatic flame temperatures for Series A as a function of fuel concentration for various oxygen levels. The figure legend lists the curves in the order in which they appear in the graph. For example, the first entry in the figure legend (isoprene in air at 40° C.) corresponds to the highest curve in the graph.

FIG. 69 is a graph of the calculated adiabatic flame temperatures for Series B as a function of fuel concentration for various oxygen levels with 4% water. The figure legend lists the curves in the order in which they appear in the graph.

FIG. 70 is a graph of the calculated adiabatic flame temperatures for Series C as a function of fuel concentration for various oxygen levels with 5% CO₂. The figure legend lists the curves in the order in which they appear in the graph.

FIG. 71 is a graph of the calculated adiabatic flame temperatures for Series D as a function of fuel concentration for various oxygen levels with 10% CO₂. The figure legend lists the curves in the order in which they appear in the graph.

FIG. 72 is a graph of the calculated adiabatic flame temperatures for Series E as a function of fuel concentration for various oxygen levels with 15% CO₂. The figure legend lists the curves in the order in which they appear in the graph.

FIG. 73 is a graph of the calculated adiabatic flame temperatures for Series F as a function of fuel concentration for various oxygen levels with 20% CO₂. The figure legend lists the curves in the order in which they appear in the graph.

FIG. 74 is a graph of the calculated adiabatic flame temperatures for Series G as a function of fuel concentration for various oxygen levels with 30% CO₂. The figure legend lists the curves in the order in which they appear in the graph.

FIG. 75A is a table of the conversion of the CAFT Model results from weight percent to volume percent for series A.

FIG. 75B is a graph of the flammability results from the CAFT model for Series A in FIG. 68 plotted as volume percent.

FIG. 76A is a table of the conversion of the CAFT Model results from weight percent to volume percent for series B.

FIG. 76B is a graph of the flammability results from the CAFT model for Series B in FIG. 69 plotted as volume percent.

FIG. 77 is a figure depicting the flammability test vessel.

FIG. 78A is a graph of the flammability Curve for Test Series 1: 0% Steam, 0 psig, and 40° C.

FIG. 78B is a table summarizing the explosion and non-explosion data points for Test Series 1.

FIG. 78C is a graph of the flammability curve for Test Series 1 compared with the CAFT Model.

FIG. 79A is a graph of the flammability curve for Test Series 2: 4% Steam, 0 psig, and 40° C.

FIG. 79B is a table summarizing the explosion and non-explosion data points for Test Series 2.

FIG. 79C is a graph of the flammability curve for Test Series 2 compared with the CAFT Model.

FIGS. 80A-B are a table of the detailed experimental conditions and results for Test Series 1.

FIG. 81 is a table of the detailed experimental conditions and results for Test Series 2.

FIG. 82 is a graph of the calculated adiabatic flame temperature plotted as a function of fuel concentration for various nitrogen/oxygen ratios at 3 atmospheres of pressure.

FIG. 83 is a graph of the calculated adiabatic flame temperature plotted as a function of fuel concentration for various nitrogen/oxygen ratios at 1 atmosphere of pressure.

FIG. 84 is a graph of the flammability envelope constructed using data from FIG. 82 and following the methodology described in Example 13. The experimental data points (circles) are from tests described herein that were conducted at 1 atmosphere initial system pressure.

FIG. 85 is a graph of the flammability envelope constructed using data from FIG. 83 and following the methodology described in Example 13. The experimental data points (circles) are from tests described herein that were conducted at 1 atmosphere initial system pressure.

FIG. 86A is a GC/MS chromatogram of fermentation off-gas.

FIG. 86B is an expansion of FIG. 86A to show minor volatiles present in fermentation off-gas.

FIG. 87A is a GC/MS chromatogram of trace volatiles present in off-gas following cryo-trapping at −78° C.

FIG. 87B is a GC/MS chromatogram of trace volatiles present in off-gas following cryo-trapping at −196° C.

FIG. 87C is an expansion of FIG. 87B.

FIG. 87D is an expansion of FIG. 87C.

FIGS. 88A-B are GC/MS chromatogram comparing C5 hydrocarbons from petroleum-derived isoprene (FIG. 88A) and biologically produced isoprene (FIG. 88B). The standard contains three C5 hydrocarbon impurities eluting around the main isoprene peak (FIG. 88A). In contrast, biologically produced isoprene contains amounts of ethanol and acetone (run time of 3.41 minutes) (FIG. 88A).

FIG. 89 is a graph of the analysis of fermentation off-gas of an E. coli BL21 (DE3) pTrcIS strain expressing a Kudzu isoprene synthase and fed glucose with 3 g/L yeast extract.

FIG. 90 shows the structures of several impurities that are structurally similar to isoprene and may also act as polymerization catalyst poisons.

FIG. 91 is a map of pTrcHis2AUpperPathway (also called pTrcUpperMVA).

FIGS. 92A-92C are the nucleotide sequence of pTrcHis2AUpperPathway (also called pTrcUpperMVA) (SEQ ID NO:23).

FIG. 93 is a time course of optical density within the 15-L bioreactor fed with glucose.

FIG. 94 is a time course of isoprene titer within the 15-L bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

FIG. 95 is a time course of total isoprene produced from the 15-L bioreactor fed with glucose.

FIG. 96 is a time course of optical density within the 15-L bioreactor fed with invert sugar.

FIG. 97 is a time course of isoprene titer within the 15-L bioreactor fed with invert sugar. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

FIG. 98 is a time course of total isoprene produced from the 15-L bioreactor fed with invert sugar.

FIG. 99 is a time course of optical density within the 15-L bioreactor fed with glucose.

FIG. 100 is a time course of isoprene titer within the 15-L bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

FIG. 101 is a time course of isoprene specific activity from the 15-L bioreactor fed with glucose.

FIG. 102 is a map of pCLPtrcUpperPathwayHGS2.

FIGS. 103A-103C are the nucleotide sequence of pCLPtrcUpperPathwayHGS2 (SEQ ID NO:24).

FIG. 104 is a time course of optical density within the 15-L bioreactor fed with glucose.

FIG. 105 is a time course of isoprene titer within the 15-L bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

FIG. 106 is a time course of total isoprene produced from the 15-L bioreactor fed with glucose.

FIG. 107 is a map of plasmid MCM330 (FRT-cm-FRT-gi1.2-KKDy at attTn7).

FIGS. 108A-108C are the nucleotide sequence of plasmid MCM330 (SEQ ID NO:25).

FIG. 109 is a map of pET24D-Kudzu.

FIGS. 110A-B are the nucleotide sequence of pET24D-Kudzu (SEQ ID NO:26).

FIG. 111A is a time course of optical density within the 15-L bioreactor fed with glucose.

FIG. 111B is a time course of isoprene titer within the 15-L bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

FIG. 111C is a time course of specific productivity of isoprene in the 15-L bioreactor fed with glucose.

FIG. 112A is a map of the M. mazei archaeal Lower Pathway operon.

FIGS. 112B-C are the nucleotide sequence of the M. mazei archaeal lower Pathway operon (SEQ ID NO:27).

FIG. 113A is a map of MCM382-pTrcKudzuMVK(mazei).

FIGS. 113B-C are the nucleotide sequence of MCM382-pTrcKudzuMVK(mazei) (SEQ ID NO:28).

FIG. 114A is a map of MCM376-MVK from M. mazei archaeal Lower in pET200D.

FIGS. 114B-C are the nucleotide sequence of MCM376-MVK from M. mazei archaeal Lower in pET200D (SEQ ID NO:29).

FIGS. 115A-115D demonstrate that over-expression of MVK and isoprene synthase results in increased isoprene production. Accumulated isoprene and CO₂ from MCM401 and MCM343 during growth on glucose in 100 mL bioreactors with 100 and 200 μM IPTG induction of isoprene production was measured over a 22 hour time course. FIG. 115A is a graph of the accumulated isoprene (%) from MCM343. FIG. 115B is a graph of the accumulated isoprene (%) from MCM401. FIG. 115C is a graph of the accumulated CO₂(%) from MCM343. FIG. 115D is a graph of the accumulated CO₂(%) from MCM401.

FIG. 116 is a time course of optical density within the 15-L bioreactor fed with glucose.

FIG. 117 is a time course of isoprene titer within the 15-L bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

FIG. 118 is a time course of total isoprene produced from the 15-L bioreactor fed with glucose.

FIG. 119 is a graph of the total carbon dioxide evolution rate (TCER), or metabolic activity profile, within the 15-L bioreactor fed with glucose.

FIG. 120 is a graph of the cell viability during isoprene production within the 15-L bioreactor fed with glucose. TVC/OD is the total viable counts (colony forming units) in 1 mL of broth per optical density unit (OD₅₅₀).

FIG. 121 is a time course of optical density within the 15-L bioreactor fed with glucose.

FIG. 122 is a time course of isoprene titer within the 15-L bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

FIG. 123 is a time course of total isoprene produced from the 15-L bioreactor fed with glucose.

FIG. 124 is a time course of volumetric productivity within the 15-L bioreactor fed with glucose. The volumetric productivity is defined as the amount of isoprene produced per liter of broth per hour.

FIG. 125 is a time course of instantaneous yield within the 15-L bioreactor fed with glucose. The instantaneous yield is defined as the amount of isoprene (gram) produced per amount of glucose (gram) fed to the bioreactor (w/w) during the time interval between the data points.

FIG. 126 is a graph of the total carbon dioxide evolution rate (TCER), or metabolic activity profile, within the 15-L bioreactor fed with glucose.

FIG. 127 is cell viability during isoprene production within the 15-L bioreactor fed with glucose. TVC/OD is the total viable counts (colony forming units) in 1 mL of broth per optical density unit (OD₅₅₀).

FIG. 128 is a time course of optical density within the 15-L bioreactor fed with glucose.

FIG. 129 is a time course of isoprene titer within the 15-L bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

FIG. 130 is a time course of total isoprene produced from the 15-L bioreactor fed with glucose.

FIG. 131 is a graph of total carbon dioxide evolution rate (TCER), or metabolic activity profile, within the 15-L bioreactor fed with glucose.

FIG. 132 is a graph showing that a transient decrease in the airflow to the bioreactor caused a spike in the concentration of isoprene in the off-gas that did not cause a dramatic decrease in metabolic activity (TCER). TCER, or metabolic activity, is the total carbon dioxide evolution rate.

FIG. 133 is a graph of the cell viability during isoprene production within the 15-L bioreactor fed with glucose. TVC/OD is the total viable counts (colony forming units) in 1 mL of broth per optical density unit (OD₅₅₀).

FIG. 134 is a time course of optical density within the 15-L bioreactor fed with glucose. Dotted vertical lines denote the time interval when isoprene was introduced into the bioreactor at a rate of 1 g/L/hr.

FIG. 135 is total carbon dioxide evolution rate (TCER), or metabolic activity profile, within the 15-L bioreactor fed with glucose. Dotted vertical lines denote the time interval when isoprene was introduced into the bioreactor at a rate of 1 g/L/hr.

FIG. 136 is cell viability during isoprene production within the 15-L bioreactor fed with glucose. TVC/OD is the total viable counts (colony forming units) in 1 mL of broth per optical density unit (OD₅₅₀). Dotted vertical lines denote the time interval when isoprene was introduced into the bioreactor at a rate of 1 g/L/hr.

FIGS. 137A-B are the sequence of Populus alba pET24a: isoprene synthase gene highlighted in bold letters (SEQ ID NO:30).

FIGS. 137C-D are the sequence of Populus nigra pET24a: isoprene synthase gene highlighted in bold letters (SEQ ID NO:31).

FIGS. 137E-F are the sequence of Populus tremuloides pET24a (SEQ ID NO:32).

FIG. 137G is the amino acid sequence of Populus tremuloides isoprene synthase gene (SEQ ID NO:33).

FIGS. 137H-I are the sequence of Populus trichocarpa pET24a: isoprene synthase gene highlighted in bold letters (SEQ ID NO:34).

FIGS. 137J-K are the sequence of Populus tremula x Populus alba pET24a: isoprene synthase gene highlighted in bold letters (SEQ ID NO:35).

FIG. 137L is a map of MCM93 which contains the kudzu IspS coding sequence in a pCR2.1 backbone.

FIGS. 137M-N are the sequence of MCM93 (SEQ ID NO:36).

FIG. 137O is a map of pET24D-Kudzu.

FIGS. 137P-Q are the sequence of pET24D-Kudzu (SEQ ID NO:37).

FIG. 138 is isoprene synthase expression data for various poplar species as measured in the whole cell head space assay. Y-axis is μg/L/OD of isoprene produced by 0.2 mL of a culture induced with IPTG.

FIG. 139 is relative activity of Poplar isoprene synthase enzymes as measured by DMAPP assay. Poplar enzymes have significantly higher activity than the isoprene synthase from Kudzu. Poplar [alba x tremula] only had traces (<1%) of activity and is not shown in the plot.

FIG. 140 is a map of pDONR221:19430-hybrid_HGS.

FIG. 141 is the nucleotide sequence of pDONR221:19430-hybrid_HGS, the sequence of Kudzu isoprene synthase codon-optimized for yeast (SEQ ID NO:38).

FIG. 142A is a map of pDW14.

FIGS. 142B-C are the complete nucleotide sequence of pDW14 (SEQ ID NO:39).

FIG. 143 shows induced INVSc-1 strains harboring pDW14 or pYES-DEST52. FIG. 143A. A 4-12% bis tris gel (Novex, Invitrogen) of lysates generated from INVSc-1 strains induced with galactose and stained with SimplyBlue SafeStain (Invitrogen). FIG. 143B. Western blot analysis of the same strains using the WesternBreeze kit (Invitrogen). Lanes are as follows: 1, INVSc-1+pYES-DEST52; 2, INVSc-1+pDW14 (isolate 1); 3, INVSc-1+pDW14 (isolate 2). MW (in kDa) is indicated (using the SeeBlue Plus2 molecular weight standard).

FIG. 144 (FIGS. 144A and 144B) shows induced INVSc-1 strains harboring pDW14 or pYES-DEST52. FIG. 144A. OD₆₀₀ of galactose-induced strains prior to lysis. The y-axis is OD₆₀₀. FIG. 144B. DMAPP assay of isoprene synthase headspace in control and isoprene synthase-harboring strains. Specific activity was calculated as μg HG/L/OD. Samples are as follows: Control, INVSc-1+pYES-DEST52; HGS-1, INVSc-1+pDW14 (isolate 1); HGS-2, INVSc-1+pDW14 (isolate 2).

FIG. 145A is a map of codon optimized isoprene synthase fluo-opt2v2.

FIG. 145B is the nucleotide sequence of codon optimized isoprene synthase fluo-opt2v2 (SEQ ID NO:40).

FIG. 146A is a map of pBBR1MCS5.

FIGS. 146B-C are the nucleotide sequence of pBBR1MCS5 (SEQ ID NO:41).

FIG. 147A is a map of pBBR5HGSOpt2_(—)2.

FIGS. 147B-C are the nucleotide sequence of pBBR5HGSOpt2_(—)2 (SEQ ID NO:42).

FIG. 148 is a graph of CER versus fermentation time for strain MCM401, uninduced, induced with IPTG (4×50 μmol) or IPTG (2×100 μmol).

FIG. 149 shows concentration of glucose in sugar cane solutions, pH adjusted or not, as a function of the number of autoclaving cycles (one cycle=30 min).

FIG. 150 shows growth curves (OD₆₀₀ as a function of time) of Pseudomonas putida F1 and Pseudomonas fluorescens ATCC13525 on glucose, sugar cane, and inverted sugar cane.

FIG. 151 shows growth curves (OD₆₀₀ as a function of time) of E. coli BL21(DE3), MG1655, ATCC11303 and B REL 606 on glucose, sugar cane, and inverted sugar cane.

FIG. 152 is a map of plasmid pET24 P. alba HGS.

FIG. 153A-B are the nucleotide sequence of plasmid pET24 P. alba HGS (SEQ ID NO:43).

FIG. 154 is a schematic diagram showing restriction sites used for endonuclease digestion to construct plasmid EWL230 and compatible cohesive ends between BspHI and NcoI sites.

FIG. 155 is a map of plasmid EWL230.

FIGS. 156A-B are the nucleotide sequence of plasmid EWL230 (SEQ ID NO:44).

FIG. 157 is a schematic diagram showing restriction sites used for endonuclease digestion to construct plasmid EWL244 and compatible cohesive ends between NsiI and PstI sites.

FIG. 158 is a map of plasmid EWL244.

FIGS. 159A-B are the nucleotide sequence of plasmid EWL244 (SEQ ID NO:45).

FIG. 160A is a map of the M. mazei archaeal Lower Pathway operon.

FIGS. 160B-C are the nucleotide sequence of the M. mazei archaeal Lower Pathway operon (SEQ ID NO:46).

FIG. 161A is a map of MCM376-MVK from M. mazei archaeal Lower in pET200D.

FIGS. 161B-C are the nucleotide sequence of MCM376-MVK from M. mazei archaeal Lower in pET200D (SEQ ID NO:47).

FIG. 162 is a map of plasmid pBBRCMPGI1.5-pgl.

FIGS. 163A-B are the nucleotide sequence of plasmid pBBRCMPGI1.5-pgl (SEQ ID NO:48).

FIGS. 164A-F are graphs of isoprene production by E. coli strain expressing M. mazei mevalonate kinase, P. alba isoprene synthase, and pgl (RHM111608-2), and grown in fed-batch culture at the 15-L scale. FIG. 164A shows the time course of optical density within the 15-L bioreactor fed with glucose. FIG. 164B shows the time course of isoprene titer within the 15-L bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth. Method for calculating isoprene: cumulative isoprene produced in 59 hrs, g/Fermentor volume at 59 hrs, L [=] g/L broth. FIG. 164C also shows the time course of isoprene titer within the 15-L bioreactor fed with glucose. Method for calculating isoprene: ∫(Instantaneous isoprene production rate, g/L/hr)dt from t=0 to 59 hours [=] g/L broth. FIG. 164D shows the time course of total isoprene produced from the 15-L bioreactor fed with glucose. FIG. 164E shows volumetric productivity within the 15-L bioreactor fed with glucose. FIG. 164F shows carbon dioxide evolution rate (CER), or metabolic activity profile, within the 15-L bioreactor fed with glucose.

FIGS. 165A-B are graphs showing analysis of off-gas from fermentation in 15 L bioreactors. Sample A is strain RM111608-2 sampled at 64.8 hours. Sample B is strain EWL256 was E. coli BL21 (DE3), pCL upper, cmR-gi1.2-yKKDyI, pTrcAlba-mMVK sampled at 34.5 hours. Hydrogen is detected above the baseline (0.95×10⁻⁸ ton) for both samples.

FIG. 166A shows an exemplary Bioisoprene™ recovery unit.

FIG. 166B shows an exemplary Bioisoprene™ desorption/condensation setup.

FIG. 167 shows a GC/FID chromatogram of a Bioisoprene™ product. The material was determined to be 99.7% pure.

FIG. 168A-C show the GC/FID chromatograms of a Bioisoprene™ sample before (A) and after treatment with alumina (B) or silica (C). The isoprene peak is not shown in these chromatograms.

FIG. 169 shows a diagram of a process and associated apparatus for purifying isoprene from a fermentation off-gas.

FIG. 170 shows GC/FID chromatogram of partially hydrogenated BioIsoprene™ monomer. Compound 1 (RT=12.30 min)=3-methyl-1-butene, compound 2 (RT=12.70 min)=2-methylbutane, compound 3 (RT=13.23 min)=2-methyl-1-butene, compound 4 (RT=13.53 min)=isoprene, compound 5 (RT=14.01 min)=2-methyl-2-butene).

FIG. 171 shows the GC/MS Total Ion Chromatogram for products derived from the Amberlyst-15 acid resin-catalyzed dimerization of 2-methyl-2-butene.

FIG. 172 shows the GC/MS Total Ion Chramatogram for products derived from Amberlyst 15 acid resin-catalyzed oligomerization of BioIsoprene™ monomer.

FIG. 173 shows a process flow diagram for the conversion of a C5 stream into a C10/C15 product stream using a dimerization reactor. The C5 stream comprises BioIsoprene™ monomer and/or C5 derivatives of BioIsoprene™ monomer.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides, inter alia, compositions and methods for producing a fuel constituent from isoprene. Provided herein are fuel constituents or additives, for example, cyclic isoprene dimers and trimers, linear isoprene oligomers, aromatic and alicyclic isoprene derivatives, and oxygenated isoprene derivatives. The fuel constituent can be produced by chemical transformations of a starting material comprising a commercially beneficial amount of highly pure isoprene. In one aspect, the commercially beneficial amount of highly pure isoprene comprises bioisoprene. In another aspect, a commercially beneficial amount of highly pure isoprene can be bioisoprene. In another aspect, a commercially beneficial amount of highly pure isoprene can be highly pure isoprene compositions produced by culturing cells expressing a heterologous isoprene synthase enzyme. In other aspects, highly pure isoprene undergoes oligomerization to form unsaturated isoprene oligomers such as cyclic dimers or trimers and linear oligomers. The unsaturated oligomers may be hydrogenated to produce saturated hydrocarbon fuel constituent. In some embodiment, reaction of highly pure isoprene with alcohols in the presence of an acid catalyst produces fuel oxygenates. In another aspect, the highly pure isoprene is partially hydrogenated to produce isoamylenes. In some embodiments, an isoamylene product derived from the highly pure isoprene undergoes dimerization to form isodecenes. In some embodiments, isoamylene products derived from the highly pure isoprene react with alcohols in the presence of an acid catalyst to produce fuel oxygenates.

Bioisoprene derived from renewable carbon can be converted to a variety of hydrocarbon fuels by chemical catalysis. Provided herein are methods for recovering isoprene from fermentation and subsequent conversion to hydrocarbon fuels by chemical catalysis to compounds of higher molecular weight. These methods include, but are not limited to, recovering and purifying isoprene from fermentation off-gas and subsequent gas or liquid phase catalysis to provide compounds with fuel value. Both continuous and batch mode processes are contemplated within the scope of the invention.

As further detailed herein, a bioisoprene composition is distinguished from a petro-isoprene composition in that a bioisoprene composition is substantially free of any contaminating unsaturated C5 hydrocarbons that are usually present in petro-isoprene compositions, such as, but not limited to, 1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne. If any contaminating unsaturated C5 hydrocarbons are present in the bioisoprene starting material described herein, they are present in lower levels than that in petro-isoprene compositions. Accordingly, any fuel products derived from bioisoprene compositions described herein is essentially free of, or contains at lower levels than that in fuel products derived from petro-isoprene, any contaminating unsaturated C5 hydrocarbons or products derived from such contaminating unsaturated C5 hydrocarbons. In addition, the sulfur levels in a bioisoprene composition are lower than the sulfur levels in petro-isoprene compositions. Fuels products derived from bioisoprene compositions contain lower levels of sulfur than that in fuel products derived from petro-isoprene.

Bioisoprene is distinguished from petro-isoprene in that bioisoprene is produced with other bio-byproducts (compounds derived from the biological sources and/or associated the biological processes that are obtained together with bioisoprene) that are not present or present in much lower levels in petro-isoprene compositions, such as alcohols, aldehydes, ketone and the like. The bio-byproducts may include, but are not limited to, ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or a linear isoprene polymer (such as a linear isoprene dimer or a linear isoprene trimer derived from the polymerization of multiple isoprene units). Fuel products derived from bioisoprene contain one or more of the bio-byproducts or compounds derived from any of the bio-byproducts. In addition, fuel products derived from bioisoprene may contain compounds formed from these bio-byproducts during subsequent chemical conversion. Examples of such compounds include those derived from Diels-Alder cycloaddition of dienophiles to isoprene or fuel derivatives thereof, the oxidation of isoprene or fuel derivatives.

Further, bioisoprene is distinguished from petro-isoprene by carbon finger-printing. In one aspect, bioisoprene has a higher radioactive carbon-14 (¹⁴C) content or higher ¹⁴C/¹²C ratio than petro-isoprene. Bioisoprene is produced from renewable carbon sources, thus the ¹⁴C content or the ¹⁴C/¹²C ratio in bioisoprene is the same as that in the present atmosphere. Petro-isoprene, on the other hand, is derived from fossil fuels deposited thousands to millions of years ago, thus the ¹⁴C content or the ¹⁴C/¹²C ratio is diminished due to radioactive decay. As discussed in greater detail herein, the fuel products derived from bioisoprene has higher ¹⁴C content or ¹⁴C/¹²C ratio than fuel products derived from petro-isoprene. In one embodiment, a fuel product derived from bioisoprene described herein has a ¹⁴C content or ¹⁴C/¹²C ratio similar to that in the atmosphere. In another aspect, bioisoprene can be analytically distinguished from petro-isoprene by the stable carbon isotope ration (¹³C/¹²C), which can be reported as “delta values” represented by the symbol d¹³C. For examples, for isoprene derived from extractive distillation of C₅ streams from petroleum refineries, δ¹³C is about −22‰ to about −24‰. This range is typical for light, unsaturated hydrocarbons derived from petroleum, and products derived from petroleum-based isoprene typically contain isoprenic units with the same δ¹³C. Bioisoprene produced by fermentation of corn-derived glucose (δ¹³C −10.73‰) with minimal amounts of other carbon-containing nutrients (e.g., yeast extract) produces isoprene which can be polymerized into polyisoprene with δ¹³C −14.66‰ to −14.85‰. Products produced from such bioisoprene are expected to have δ¹³C values that are less negative than those derived from petroleum-based isoprene.

Compounds made by these methods include cyclic isoprene dimers and trimers, linear oligomers, aromatic and alicyclic derivatives. Diisoamylenes are made by methods comprising partial hydrogenation of bioisoprene compositions. These chemical derivatives of isoprene are useful as liquid transportation fuels (IsoFuels™) and as fuel additives.

Also provided herein are methods for the production of oxygenated derivatives of isoprene including alcohols, ketones, esters and ethers. Methods for the synthesis of oxygenated derivatives of isoprene can also be performed in liquid or gas phase, using homogeneous and heterogeneous catalysts. Compounds of this chemical class are also useful as liquid transportation fuels, and can be used in fuel blends as fuel oxygenates for emissions reduction and as fuel modifiers, for example as cetane boosters for diesel.

While isoprene can be obtained by fractionating petroleum, the purification of this material is expensive and time-consuming. Petroleum cracking of the C5 stream of hydrocarbons produces only about 15% isoprene. Isoprene is also naturally produced by a variety of microbial, plant, and animal species. In particular, two pathways have been identified for the biosynthesis of isoprene: the mevalonate (MVA) pathway and the non-mevalonate (DXP) pathway. Genetically engineered cell cultures in bioreactors have produced isoprene more efficiently, in larger quantities, in higher purities and/or with unique impurity profiles, e.g. as described in U.S. provisional patent application Nos. 61/013,386 and 61/013,574, filed on Dec. 13, 2007, WO 2009/076676, U.S. provisional patent application Nos. 61/134,094, 61/134,947, 61/134,011 and 61/134,103, filed on Jul. 2, 2008, WO 2010/003007, U.S. provisional patent application No. 61/097,163, filed on Sep. 15, 2008, WO 2010/031079, U.S. provisional patent application No. 61/097,186, filed on Sep. 15, 2008, WO 2010/031062, U.S. provisional patent application No. 61/097,189, filed on Sep. 15, 2008, WO 2010/031077, U.S. provisional patent application No. 61/097,200, filed on Sep. 15, 2008, WO 2010/031068, U.S. provisional patent application No. 61/097,204, filed on Sep. 15, 2008, WO 2010/031076, U.S. provisional patent application No. 61/141,652, filed on Dec. 30, 2008, PCT/US09/069,862, U.S. patent application Ser. No. 12/335,071, filed Dec. 15, 2008 (US 2009/0203102 A1) and U.S. patent application Ser. No. 12/429,143, filed Apr. 23, 2009 (US 2010/0003716 A1), which are incorporated by reference in their entireties.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. All documents cited are, in relevant part, incorporated herein by reference. However, the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.

As used herein, the singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise.

It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

The term “isoprene” refers to 2-methyl-1,3-butadiene (CAS#78-79-5), which is the direct and final volatile C5 hydrocarbon product from the elimination of pyrophosphate from 3,3-dimethylallyl pyrophosphate (DMAPP), and does not involve the linking or polymerization of [an] IPP molecule(s) to [a] DMAPP molecule(s). The term “isoprene” is not generally intended to be limited to its method of production unless indicated otherwise herein.

As used herein, “biologically produced isoprene” or “bioisoprene” is isoprene produced by any biological means, such as produced by genetically engineered cell cultures, natural microbials, plants or animals.

A “bioisoprene composition” refers to a composition that can be produced by any biological means, such as systems (e.g., cells) that are engineered to produce isoprene. It contains isoprene and other compounds that are co-produced (including impurities) and/or isolated together with isoprene. A bioisoprene composition usually contains fewer hydrocarbon impurities than isoprene produced from petrochemical sources and often requires minimal treatment in order to be of polymerization grade. As detailed herein, bioisoprene composition also has a different impurity profile from a petrochemically produced isoprene composition.

As used herein, “at least a portion of the isoprene starting composition” can refer to at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% of the isoprene starting composition undergoing chemical transformation.

As used herein, IsoFuels™ refers to fuels including liquid transportation fuels that are derived from isoprene. BioIsoFuels™ refers to fuels including liquid transportation fuels that are derived from bioisoprene.

The term “oligomerization” as used herein refers to a chemical process for combining two or more monomer units. “Oligomerization” of isoprene produces a derivative of isoprene derived from two or more molecules of isoprene, such as linear dimers of isoprene, cyclic dimers of isoprene, linear trimers of isoprene, cyclic trimers of isoprene and the like.

“Complete hydrogenation”, “Completely hydrogenate” or “Fully hydrogenate” is defined as the addition of hydrogen (H₂), typically in the presence of a hydrogenation catalyst, to all unsaturated functional groups, such as carbon-carbon double bonds, within a precursor compound to give fully saturated product compounds. For example, complete hydrogenation of isoprene forms isopentane whereby 2 moles of H₂ is consumed per mole of isoprene.

“Partial hydrogenation” or “Partially hydrogenate” is defined as the addition of hydrogen (H₂), typically in the presence of a hydrogenation catalyst, to at least one, but not all unsaturated functional groups, such as carbon-carbon double bonds, within a precursor compound. The product(s) of partial hydrogenation can be further completely hydrogenated to give fully saturated product compounds. Partial hydrogenation of a diene forms one or more mono-olefins. For example, partial hydrogenation of isoprene can give 3 isomeric isopentenes (2-methylbut-1-ene, 2-methylbut-2-ene and 3-methylbut-1-ene) whereby 1 mole of H₂ is consumed per mole of isoprene.

“Selective hydrogenation” or “Selectively hydrogenate” is defined as the addition of hydrogen (H₂), typically in the presence of a hydrogenation catalyst, to at least one, but not all unsaturated functional groups, such as carbon-carbon double bonds, within a precursor compound whereby certain unsaturated functional groups are preferentially hydrogenated over other unsaturated groups under the chosen conditions. For example, selective hydrogenation of isoprene may form preferentially 2-methyl-2-butene, 2-methyl-1-butene, 3-methyl-1-butene or a mixture thereof.

As used herein, the term “polypeptides” includes polypeptides, proteins, peptides, fragments of polypeptides, and fusion polypeptides.

As used herein, an “isolated polypeptide” is not part of a library of polypeptides, such as a library of 2, 5, 10, 20, 50 or more different polypeptides and is separated from at least one component with which it occurs in nature. An isolated polypeptide can be obtained, for example, by expression of a recombinant nucleic acid encoding the polypeptide.

By “heterologous polypeptide” is meant a polypeptide whose amino acid sequence is not identical to that of another polypeptide naturally expressed in the same host cell. In particular, a heterologous polypeptide is not identical to a wild-type polypeptide that is found in the same host cell in nature.

“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid for improved expression in a host cell, it is desirable in some embodiments to design the nucleic acid such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

As used herein, a “nucleic acid” refers to two or more deoxyribonucleotides and/or ribonucleotides covalently joined together in either single or double-stranded form.

By “recombinant nucleic acid” is meant a nucleic acid of interest that is free of one or more nucleic acids (e.g., genes) which, in the genome occurring in nature of the organism from which the nucleic acid of interest is derived, flank the nucleic acid of interest. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA, a genomic DNA fragment, or a cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.

By “heterologous nucleic acid” is meant a nucleic acid whose nucleic acid sequence is not identical to that of another nucleic acid naturally found in the same host cell. In particular, a heterologous nucleic acid is not identical to a wild-type nucleic acid that is found in the same host cell in nature.

As used herein, a “vector” means a construct that is capable of delivering, and desirably expressing one or more nucleic acids of interest in a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, DNA or RNA expression vectors, cosmids, and phage vectors.

As used herein, an “expression control sequence” means a nucleic acid sequence that directs transcription of a nucleic acid of interest. An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. An “inducible promoter” is a promoter that is active under environmental or developmental regulation. The expression control sequence is operably linked to the nucleic acid segment to be transcribed.

The term “selective marker” or “selectable marker” refers to a nucleic acid capable of expression in a host cell that allows for ease of selection of those host cells containing an introduced nucleic acid or vector. Examples of selectable markers include, but are not limited to, antibiotic resistance nucleic acids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/or nucleic acids that confer a metabolic advantage, such as a nutritional advantage on the host cell. Exemplary nutritional selective markers include those markers known in the art as amdS, argB, and pyr4.

Compositions and Systems

Isoprene derived from petrochemical sources usually is an impure C5 hydrocarbon fraction which requires extensive purification before the material is suitable for polymerization or other chemical transformations. Several impurities are particularly problematic given their structural similarity to isoprene and the fact that they can act as polymerization catalyst poisons. Such compounds include, but are not limited to, 1,3-cyclopentadiene, cis- and trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne. As detailed below, biologically produced isoprene can be substantially free of any contaminating unsaturated C5 hydrocarbons without undergoing extensive purification. Some biologically produced isoprene compositions contain ethanol, acetone, and C5 prenyl alcohols. These components are more readily removed from the isoprene stream than the isomeric C5 hydrocarbon fractions that are present in isoprene compositions derived from petrochemical sources. Further, these impurities can be managed in the bioprocess, for example by genetic modification of the producing strain, carbon feedstock, alternative fermentation conditions, recovery process modifications and additional or alternative purification methods.

In one aspect, the invention features compositions and systems for producing a fuel constituent from isoprene comprising: (a) a commercially beneficial amount of highly pure isoprene starting composition; and (b) a fuel constituent produced from at least a portion of the highly pure isoprene starting material; where at least a portion of the commercially beneficial amount of highly pure isoprene starting composition undergoes a chemical transformation. A highly pure isoprene starting material is subjected to chemical reactions to produce a commercially beneficial amount of product that is useful for making fuels. In one aspect, a commercially beneficial amount of highly pure isoprene comprises bioisoprene. In one aspect, a commercially beneficial amount of highly pure isoprene can be bioisoprene.

Exemplary Starting Isoprene Compositions

In some embodiments, the commercially beneficial amount of highly pure isoprene starting composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene. In some embodiments, the starting isoprene composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene. In some embodiments, the starting isoprene composition comprises greater than or about 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 kg of isoprene. In some embodiments, the amount of isoprene in the starting composition is between about 2 to about 5,000 mg, such as between about 2 to about 100 mg, about 100 to about 500 mg, about 500 to about 1,000 mg, about 1,000 to about 2,000 mg, or about 2,000 to about 5,000 mg. In some embodiments, the amount of isoprene in the starting composition is between about 20 to about 5,000 mg, about 100 to about 5,000 mg, about 200 to about 2,000 mg, about 200 to about 1,000 mg, about 300 to about 1,000 mg, or about 400 to about 1,000 mg. In some embodiments, the amount of isoprene in the starting composition is between about 2 to about 5,000 g, such as between about 2 to about 100 g, about 100 to about 500 g, about 500 to about 1,000 g, about 1,000 to about 2,000 g, or about 2,000 to about 5,000 g. In some embodiments, the amount of isoprene in the starting composition is between about 2 to about 5,000 kg, about 10 to about 2,000 kg, about 20 to about 1,000 kg, about 20 to about 500 kg, about 30 to about 200 kg, or about 40 to about 100 kg. In some embodiments, greater than or about 20, 25, 30, 40, 50, 60, 70, 80, 90, or 95% (w/w) of the volatile organic fraction of the starting composition is isoprene.

In some embodiments, the highly pure isoprene starting composition comprises greater than or about 98.0, 98.5, 99.0, 99.5, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the starting composition. In some embodiments, the highly pure isoprene starting composition comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the starting composition. In some embodiments, the starting composition has a relative detector response of greater than or about 98.0, 98.5, 99.0, 99.5, or 100% for isoprene compared to the detector response for all C5 hydrocarbons in the starting composition. In some embodiments, the starting composition has a relative detector response of greater than or about 99.90, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96, 99.97, 99.98, 99.99, or 100% for isoprene compared to the detector response for all C5 hydrocarbons in the starting composition. In some embodiments, the starting isoprene composition comprises between about 98.0 to about 98.5, about 98.5 to about 99.0, about 99.0 to about 99.5, about 99.5 to about 99.8, about 99.8 to 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the starting composition. In some embodiments, the starting isoprene composition comprises between about 99.90 to about 99.92, about 99.92 to about 99.94, about 99.94 to about 99.96, about 99.96 to about 99.98, about 99.98 to 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the starting composition.

In some embodiments, the highly pure isoprene starting composition comprises less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the starting composition. In some embodiments, the starting composition has a relative detector response of less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for C5 hydrocarbons other than isoprene compared to the detector response for all C5 hydrocarbons in the starting composition. In some embodiments, the starting composition has a relative detector response of less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne compared to the detector response for all C5 hydrocarbons in the starting composition. In some embodiments, the highly pure isoprene starting composition comprises between about 0.02 to about 0.04%, about 0.04 to about 0.06%, about 0.06 to 0.08%, about 0.08 to 0.10%, or about 0.10 to about 0.12% C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the starting composition.

In some embodiments, the highly pure isoprene starting composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a compound that inhibits the polymerization of isoprene for any compound in the starting composition that inhibits the polymerization of isoprene. In some embodiments, the starting isoprene composition comprises between about 0.005 to about 50, such as about 0.01 to about 10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005 μg/L of a compound that inhibits the polymerization of isoprene for any compound in the starting composition that inhibits the polymerization of isoprene. In some embodiments, the starting isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a hydrocarbon other than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne). In some embodiments, the starting isoprene composition comprises between about 0.005 to about 50, such as about 0.01 to about 10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005 μg/L of a hydrocarbon other than isoprene. In some embodiments, the starting isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a protein or fatty acid (such as a protein or fatty acid that is naturally associated with natural rubber).

In some embodiments, the highly pure isoprene starting composition comprises less than or about 10, 5, 1, 0.8, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of alpha acetylenes, piperylenes, acetonitrile, or 1,3-cyclopentadiene. In some embodiments, the starting isoprene composition comprises less than or about 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of sulfur or allenes. In some embodiments, the starting isoprene composition comprises less than or about 30, 20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of all acetylenes (such as 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne). In some embodiments, the starting isoprene composition comprises less than or about 2000, 1000, 500, 200, 100, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of isoprene dimers, such as cyclic isoprene dimers (e.g., cyclic C10 compounds derived from the dimerization of two isoprene units).

In some embodiments, the highly pure isoprene starting composition includes ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more of the foregoing. In particular embodiments, the starting isoprene composition comprises greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more of the foregoing. In some embodiments, the isoprene composition comprises between about 0.005 to about 120, such as about 0.01 to about 80, about 0.01 to about 60, about 0.01 to about 40, about 0.01 to about 30, about 0.01 to about 20, about 0.01 to about 10, about 0.1 to about 80, about 0.1 to about 60, about 0.1 to about 40, about 5 to about 80, about 5 to about 60, or about 5 to about 40 μg/L of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol, or any two or more of the foregoing.

In some embodiments, the highly pure isoprene starting composition includes one or more of the following components: 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or a linear isoprene polymer (such as a linear isoprene dimer or a linear isoprene trimer derived from the polymerization of multiple isoprene units). In various embodiments, the amount of one of these components relative to amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w). In some embodiments, the relative detector response for the second compound compared to the detector response for isoprene is greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%. In various embodiments, the amount of one of these components relative to amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is between about 0.01 to about 105% (w/w), such as about 0.01 to about 90, about 0.01 to about 80, about 0.01 to about 50, about 0.01 to about 20, about 0.01 to about 10, about 0.02 to about 50, about 0.05 to about 50, about 0.1 to about 50, or 0.1 to about 20% (w/w).

In some embodiments, at least a portion of the highly pure isoprene starting composition is in a gas phase. In some embodiments, at least a portion of the highly pure isoprene starting composition is in a liquid phase (such as a condensate). In some embodiments, at least a portion of the highly pure isoprene starting composition is in a solid phase. In some embodiments, at least a portion of the highly pure isoprene starting composition is absorbed to a solid support, such as a support that includes silica and/or activated carbon. In some embodiments, the starting isoprene composition is mixed with one or more solvents. In some embodiments, the starting isoprene composition is mixed with one or more gases.

In other embodiments, the commercially beneficial amount of highly pure isoprene starting composition is produced by a biological process. In some preferred embodiments, the highly pure isoprene starting composition is a bioisoprene composition produced by culturing cells that produce greater than about 400 nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/g_(wcm)/hr) of isoprene. In one embodiment, the bioisoprene composition is produced by culturing cells that convert more than about 0.002% of the carbon in a cell culture medium into isoprene. In other embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide, e.g. a naturally-occurring polypeptide from a plant such as Pueraria, and (ii) is operably linked to a promoter, e.g. a T7 promoter. Other isoprene synthase polypeptides, for example, from poplar and variants of naturally-occurring as well as parent isoprene synthase, can be used to produce bioisoprene. Examples of isoprene synthase and its variants that can be used are described in U.S. application Ser. No. 12/429,143, which is incorporated herein in its entirety.

In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions. In some embodiment, the cells further comprise a heterologous nucleic acid encoding an IDI polypeptide. In some embodiments, the cells further comprise a heterologous nucleic acid encoding an MDV pathway polypeptide. In some embodiment, the starting isoprene composition is an isoprene composition as descried or is produced by culturing any of the cells described in U.S. provisional patent application Nos. 61/134,094, filed on Jul. 2, 2008, WO 2010/003007, and U.S. patent application Ser. No. 12/335,071, filed Dec. 15, 2008 (US 2009/0203102 A1), which are incorporated by reference in their entireties.

In some embodiments, the highly pure isoprene starting composition comprises a gas phase (off-gas) produced by cells in culture that produces isoprene. In some embodiments, the gas phase has a nonflammable concentration of isoprene. In some embodiments, the gas phase comprises less than about 9.5% (volume) oxygen. In some embodiments, the gas phase comprises greater than or about 9.5% (volume) oxygen, and the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit. In some embodiments, the portion of the gas phase other than isoprene comprises between about 0% to about 100% (volume) oxygen, such as between about 10% to about 100% (volume) oxygen. In some embodiments, the portion of the gas phase other than isoprene comprises between about 0% to about 99% (volume) nitrogen. In some embodiments, the portion of the gas phase other than isoprene comprises between about 1% to about 50% (volume) CO₂.

In some embodiments, the highly pure isoprene starting composition includes one or more of the following: an alcohol, an aldehyde, a ketone, or an ester (such as any of the alcohols, aldehydes, ketones or esters described herein). In some embodiments, the isoprene composition includes (i) an alcohol and an aldehyde, (ii) an alcohol and a ketone, (iii) an aldehyde and a ketone, or (iv) an alcohol, an aldehyde, and a ketone. In some embodiments, any of the isoprene compositions further includes an ester.

In some embodiments, the highly pure isoprene starting composition derived from a biological source (such as a cell culture) contains one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid, 2-butanone, 2-methyl-1-butanol, or indole. In some embodiments, the starting isoprene composition contains 1 ppm or more of one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid, 2-butanone, 2-methyl-1-butanol, or indole. In some embodiments, the concentration of more of one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid, 2-butanone, 2-methyl-1-butanol, or indole, is between about 1 to about 10,000 ppm in a starting isoprene composition (such as off-gas before it is purified). In some embodiments, the starting isoprene composition (such as off-gas after it has undergone one or more purification steps) includes one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid, 2-butanone, 2-methyl-1-butanol, or indole, at a concentration between about 1 to about 100 ppm, such as about 1 to about 10 ppm, about 10 to about 20 ppm, about 20 to about 30 ppm, about 30 to about 40 ppm, about 40 to about 50 ppm, about 50 to about 60 ppm, about 60 to about 70 ppm, about 70 to about 80 ppm, about 80 to about 90 ppm, or about 90 to about 100 ppm. In some embodiments, the starting isoprene composition contains less than 1 ppm of methanethiol (a potent catalyst poison and a source of sulfur in the final fuel product) Volatile organic compounds from cell cultures (such as volatile organic compounds in the headspace of cell cultures) can be analyzed using standard methods such as those described herein or other standard methods such as proton transfer reaction-mass spectrometry (see, for example, Bunge et al., Applied and Environmental Microbiology, 74(7):2179-2186, 2008 which is hereby incorporated by reference in its entirety, particular with respect to the analysis of volatile organic compounds).

The invention also contemplates the use of highly pure isoprene starting composition that is derived from a biological source (such as a cell culture) the co-produces isoprene and hydrogen. In some embodiments, the starting bioisoprene compositions comprise isoprene and hydrogen in ratios ranging from at least one molar percent of isoprene for every three molar percent of hydrogen to at least one molar percent of isoprene for every four molar percent of hydrogen. In some embodiments, the starting bioisoprene compositions comprise isoprene and hydrogen in molar ratios of about 1 to 9, 2 to 8, 3 to 7, 4 to 6, 5 to 5, 6 to 4, 7 to 3, 8 to 2, or 9 to 1. In some embodiments, the composition further comprises from 1 to 11 molar percent isoprene and from 4 to 44 molar percent hydrogen. In some embodiments, the composition further comprises oxygen, carbon dioxide, or nitrogen. In some embodiments, the composition further comprises from 0 to 21 molar percent oxygen, from 18 to 44 molar percent carbon dioxide, and from 0 to 78 molar percent nitrogen. In some embodiments, the composition further comprises 1.0×10⁻⁴ molar percent or less of non-methane volatile impurities. In some embodiments, the non-methane volatile impurities comprise one or more of the following: 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or a linear isoprene polymer (such as a linear isoprene dimer or a linear isoprene trimer derived from the polymerization of multiple isoprene units). In some embodiments, the non-methane volatile impurities comprise one or more of the following: the isoprene composition includes one or more of the following: an alcohol, an aldehyde, or a ketone (such as any of the alcohols, aldehydes, or ketones described herein). In some embodiments, the isoprene composition includes (i) an alcohol and an aldehyde, (ii) an alcohol and a ketone, (iii) an aldehyde and a ketone, or (iv) an alcohol, an aldehyde, and a ketone. In some embodiments, the non-methane volatile impurities comprise one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid, 2-butanone, 2-methyl-1-butanol, or indole.

Techniques for producing isoprene in cultures of cells that produce isoprene are described in U.S. provisional patent application Nos. 61/013,386 and 61/013,574, filed on Dec. 13, 2007, WO 2009/076676, U.S. provisional patent application Nos. 61/134,094, 61/134,947, 61/134,011 and 61/134,103, filed on Jul. 2, 2008, WO 2010/003007, U.S. provisional patent application No. 61/097,163, filed on Sep. 15, 2008, WO 2010/031079, U.S. provisional patent application No. 61/097,186, filed on Sep. 15, 2008, WO 2010/031062, U.S. provisional patent application No. 61/097,189, filed on Sep. 15, 2008, WO 2010/031077, U.S. provisional patent application No. 61/097,200, filed on Sep. 15, 2008, WO 2010/031068, U.S. provisional patent application No. 61/097,204, filed on Sep. 15, 2008, WO 2010/031076, U.S. provisional patent application No. 61/141,652, filed on Dec. 30, 2008, PCT/US09/069,862, U.S. patent application Ser. No. 12/335,071, filed Dec. 15, 2008 (US 2009/0203102 A1) and U.S. patent application Ser. No. 12/429,143, filed Apr. 23, 2009 (US 2010/0003716 A1), the teachings of which are incorporated herein by reference for the purpose of teaching techniques for producing and recovering isoprene by such a process. In any case, U.S. provisional patent application Nos. 61/013,386 and 61/013,574, filed on Dec. 13, 2007, WO 2009/076676, U.S. provisional patent application Nos. 61/134,094, 61/134,947, 61/134,011 and 61/134,103, filed on Jul. 2, 2008, WO 2010/003007, U.S. provisional patent application No. 61/097,163, filed on Sep. 15, 2008, WO 2010/031079, U.S. provisional patent application No. 61/097,186, filed on Sep. 15, 2008, WO 2010/031062, U.S. provisional patent application No. 61/097,189, filed on Sep. 15, 2008, WO 2010/031077, U.S. provisional patent application No. 61/097,200, filed on Sep. 15, 2008, WO 2010/031068, U.S. provisional patent application No. 61/097,204, filed on Sep. 15, 2008, WO 2010/031076, U.S. provisional patent application No. 61/141,652, filed on Dec. 30, 2008, PCT/US09/069,862, U.S. patent application Ser. No. 12/335,071, filed Dec. 15, 2008 (US 2009/0203102 A1) and U.S. patent application Ser. No. 12/429,143, filed Apr. 23, 2009 (US 2010/0003716 A1) teach compositions and methods for the production of increased amounts of isoprene in cell cultures. U.S. patent application Ser. No. 12/335,071, filed Dec. 15, 2008 and US 2009/0203102 A1 further teaches compositions and methods for co-production of isoprene and hydrogen from cultured cells. In particular, these compositions and methods compositions and methods increase the rate of isoprene production and increase the total amount of isoprene that is produced. For example, cell culture systems that generate 4.8×10⁴ nmole/g_(wcm)/hr of isoprene have been produced (Table 1). The efficiency of these systems is demonstrated by the conversion of about 2.2% of the carbon that the cells consume from a cell culture medium into isoprene. As shown in the Examples and Table 2, approximately 3 g of isoprene per liter of broth was generated. If desired, even greater amounts of isoprene can be obtained using other conditions, such as those described herein. In some embodiments, a renewable carbon source is used for the production of isoprene. In some embodiments, the production of isoprene is decoupled from the growth of the cells. In some embodiments, the concentrations of isoprene and any oxidants are within the nonflammable ranges to reduce or eliminate the risk that a fire may occur during production or recovery of isoprene. The compositions and methods are desirable because they allow high isoprene yield per cell, high carbon yield, high isoprene purity, high productivity, low energy usage, low production cost and investment, and minimal side reactions. This efficient, large scale, biosynthetic process for isoprene production provides an isoprene source for synthetic isoprene-based products such as rubber and provides a desirable, low-cost alternative to using natural rubber.

As discussed further below, the amount of isoprene produced by cells can be greatly increased by introducing a heterologous nucleic acid encoding an isoprene synthase polypeptide (e.g., a plant isoprene synthase polypeptide) into the cells. Isoprene synthase polypeptides convert dimethyl allyl diphosphate (DMAPP) into isoprene. As shown in the Examples, a heterologous Pueraria Montana (kudzu) isoprene synthase polypeptide was expressed in a variety of host cells, such as Escherichia coli, Panteoa citrea, Bacillus subtilis, Yarrowia lipolytica, and Trichoderma reesei. All of these cells produced more isoprene than the corresponding cells without the heterologous isoprene synthase polypeptide. As illustrated in Tables 1 and 2, large amounts of isoprene are produced using the methods described herein. For example, B. subtilis cells with a heterologous isoprene synthase nucleic acid produced approximately 10-fold more isoprene in a 14 liter fermentor than the corresponding control B. subtilis cells without the heterologous nucleic acid (Table 2). The production of 300 mg of isoprene per liter of broth (mg/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells) by E. coli and 30 mg/L by B. subtilis in fermentors indicates that significant amounts of isoprene can be generated (Table 2). If desired, isoprene can be produced on an even larger scale or other conditions described herein can be used to further increase the amount of isoprene. The vectors listed in Tables 1 and 2 and the experimental conditions are described in further detail below and in the Examples section.

TABLE 1 Exemplary yields of isoprene from a shake flask using the cell cultures and methods described herein. The assay for measuring isoprene production is described in Example I, part II. For this assay, a sample was removed at one or more time points from the shake flask and cultured for 30 minutes. The amount of isoprene produced in this sample was then measured. The headspace concentration and specific rate of isoprene production are listed in Table 1 and described further herein. Isoprene Production in a Headspace vial* Headspace Specific Rate concentration μg/L_(broth)/hr/OD Strain μg/L_(gas) (nmol/g_(wcm)/hr) E. coli BL21/pTrcKudzu IS 1.40 53.2 (781.2) E. coli BL21/pCL DXS yidi 7.61 289.1 (4.25 × 10³) Kudzu IS E. coli BL21/MCM127 23.0 874.1 (1.28 × 10⁴) with kudzu IS and entire MVA pathway E. coli BL21/pET 1.49 56.6 (831.1) N-HisKudzu IS Pantoea citrea/pTrcKudzu IS 0.66 25.1 (368.6) E. coli w/ Poplar IS — 5.6 (82.2) [Miller (2001)] Bacillis licheniformis Fall — 4.2 (61.4) U.S. Pat. No. 5,849,970 Yarrowia lipolytica with ~0.05 μg/L ~2 (~30) kudzu isoprene synthase Trichoderma reesei with ~0.05 μg/L ~2 (~30) kudzu isoprene synthase E. coli BL21/pTrcKKD_(y)I_(k)IS 85.9 3.2 × 10³ (4.8 × 10⁴) with kudzu IS and lower MVA pathway *Normalized to 1 mL of 1 OD₆₀₀, cultured for 1 hour in a sealed headspace vial with a liquid to headspace volume ratio of 1:19.

TABLE 2 Exemplary yields of isoprene in a fermentor using the cell cultures and methods described herein. The assay for measuring isoprene production is described in Example I, part II. For this assay, a sample of the off-gas of the fermentor was taken and analyzed for the amount of isoprene. The peak headspace concentration (which is the highest headspace concentration during the fermentation), titer (which is the cumulative, total amount of isoprene produced per liter of broth), and peak specific rate of isoprene production (which is the highest specific rate during the fermentation) are listed in Table 2 and described further herein. Isoprene Production in Fermentors Peak Peak Headspace Specific rate concentration** Titer μg/L_(broth)/hr/OD Strain (μg/L_(gas)) (mg/L_(broth)) (nmol/g_(wcm)/hr) E. coli BL21/pTrcKudzu 52 41.2 37  with Kudzu IS (543.3) E. coli FM5/pTrcKudzu 3 3.5  21.4 IS (308.1) E. coli BL21/triple strain 285 300 240   (DXS, yidi, IS) (3.52 × 10³) E. coli FM5/triple strain 50.8 29 180.8 (DXS, yidi, IS) (2.65 × 10³) E. coli/MCM127 with 3815 3044 992.5 Kudzu IS and entire (1.46 × 10⁴) MVA pathway E. coli BL21/pCLPtrc 2418 1640 1248   Upper Pathway gi1.2 (1.83 × 10⁴) integrated lower pathway pTrcKudzu E. coli BL21/MCM401 13991 23805 3733   with 4 × 50 μM IPTG (5.49 × 10⁴) E. coli BL21/MCM401 22375 19541 5839.5  with 2 × 1000 μM IPTG (8.59 × 10⁴) E. coli BL21/pCLPtrc 3500 3300 1088   UpperPathwayHGS2- (1.60 × 10⁴) pTrcKKDyIkIS Bacillus subtilis 1.5 2.5  0.8 wild-type  (11.7) Bacillus pBS Kudzu IS 16.6 ~30  5 (over  (73.4) 100 hrs) Bacillus Marburg 6051 2.04 0.61  24.5 [Wagner and Fall (1999)] (359.8) Bacillus Marburg 6051 0.7 0.15  6.8 Fall U.S. Pat. No. (100)   5,849,970 E. coli 2.03 × 10⁴ 3.22 × 10⁴  5.9 × 10³ BL21/pCLPtrcUpperPath (8.66 × 10⁴) way and gil.2KKDyI and pTrcAlba-mMVK E. coli 3.22 × 10⁴ 6.05 × 10⁴ 1.28 × 10⁴ BL21/pCLPtrcUpper (1.88 × 10⁵) Pathway and gi1.2KKDyI and pTrcAlba-mMVK plus pBBRCMPGI1.5pgl **Normalized to an off-gas flow rate of 1 vvm (1 volume off-gas per 1 L_(broth) per minute).

Additionally, isoprene production by cells that contain a heterologous isoprene synthase nucleic acid can be enhanced by increasing the amount of a 1-deoxy-D-xylulose-5-phosphate synthase (DXS) polypeptide and/or an isopentenyl diphosphate isomerase (IDI) polypeptide expressed by the cells. For example, a DXS nucleic acid and/or an IDI nucleic acid can be introduced into the cells. The DXS nucleic acid may be a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid. Similarly, the IDI nucleic acid may be a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid. In some embodiments, the amount of DXS and/or IDI polypeptide is increased by replacing the endogenous DXS and/or IDI promoters or regulatory regions with other promoters and/or regulatory regions that result in greater transcription of the DXS and/or IDI nucleic acids. In some embodiments, the cells contain both a heterologous nucleic acid encoding an isoprene synthase polypeptide (e.g., a plant isoprene synthase nucleic acid) and a duplicate copy of an endogenous nucleic acid encoding an isoprene synthase polypeptide.

The encoded DXS and IDI polypeptides are part of the DXP pathway for the biosynthesis of isoprene (FIG. 19A). DXS polypeptides convert pyruvate and D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate. While not intending to be bound by any particular theory, it is believed that increasing the amount of DXS polypeptide increases the flow of carbon through the DXP pathway, leading to greater isoprene production. IDI polypeptides catalyze the interconversion of isopentenyl diphosphate (IPP) and dimethyl allyl diphosphate (DMAPP). While not intending to be bound by any particular theory, it is believed that increasing the amount of IDI polypeptide in cells increases the amount (and conversion rate) of IPP that is converted into DMAPP, which in turn is converted into isoprene.

For example, fermentation of E. coli cells with a kudzu isoprene synthase, S. cerevisia IDI, and E. coli DXS nucleic acids was used to produce isoprene. The levels of isoprene varied from 50 to 300 μg/L over a time period of 15 hours (Example 7, part VII).

In some embodiments, the presence of heterologous or extra endogenous isoprene synthase, IDI, and DXS nucleic acids causes cells to grow more reproducibly or remain viable for longer compared to the corresponding cell with only one or two of these heterologous or extra endogenous nucleic acids. For example, cells containing heterologous isoprene synthase, IDI, and DXS nucleic acids grew better than cells with only heterologous isoprene synthase and DXS nucleic acids or with only a heterologous isoprene synthase nucleic acid. Also, heterologous isoprene synthase, IDI, and DXS nucleic acids were successfully operably linked to a strong promoter on a high copy plasmid that was maintained by E. coli cells, suggesting that large amounts of these polypeptides could be expressed in the cells without causing an excessive amount of toxicity to the cells. While not intending to be bound to a particular theory, it is believed that the presence of heterologous or extra endogenous isoprene synthase and IDI nucleic acids may reduce the amount of one or more potentially toxic intermediates that would otherwise accumulate if only a heterologous or extra endogenous DXS nucleic acid was present in the cells.

In some embodiments, the production of isoprene by cells that contain a heterologous isoprene synthase nucleic acid is augmented by increasing the amount of a MVA polypeptide expressed by the cells (FIGS. 19A and 19B). Exemplary MVA pathways polypeptides include any of the following polypeptides: acetyl-CoA acetyltransferase (AA-CoA thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK) polypeptides, phosphomevalonate kinase (PMK) polypeptides, diphosphomevalonate decarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC) polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDI polypeptides, and polypeptides (e.g., fusion polypeptides) having an activity of two or more MVA pathway polypeptides. For example, one or more MVA pathway nucleic acids can be introduced into the cells. In some embodiments, the cells contain the upper MVA pathway, which includes AA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase nucleic acids. In some embodiments, the cells contain the lower MVA pathway, which includes MVK, PMK, MVD, and IDI nucleic acids. In some embodiments, the cells contain an entire MVA pathway that includes AA-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, MVK, PMK, MVD, and IDI nucleic acids. In some embodiments, the cells contain an entire MVA pathway that includes AA-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, MVK, PMDC, IPK, and IDI nucleic acids. The MVA pathway nucleic acids may be heterologous nucleic acids or duplicate copies of endogenous nucleic acids. In some embodiments, the amount of one or more MVA pathway polypeptides is increased by replacing the endogenous promoters or regulatory regions for the MVA pathway nucleic acids with other promoters and/or regulatory regions that result in greater transcription of the MVA pathway nucleic acids. In some embodiments, the cells contain both a heterologous nucleic acid encoding an isoprene synthase polypeptide (e.g., a plant isoprene synthase nucleic acid) and a duplicate copy of an endogenous nucleic acid encoding an isoprene synthase polypeptide.

For example, E. coli cells containing a nucleic acid encoding a kudzu isoprene synthase polypeptide and nucleic acids encoding Saccharomyces cerevisiae MVK, PMK, MVD, and IDI polypeptides generated isoprene at a rate of 6.67×10⁻⁴ mol/L_(broth)/OD₆₀₀/hr (see Example 8). Additionally, a 14 liter fermentation of E. coli cells with nucleic acids encoding Enterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase polypeptides produced 22 grams of mevalonic acid (an intermediate of the MVA pathway). A shake flask of these cells produced 2-4 grams of mevalonic acid per liter. These results indicate that heterologous MVA pathways nucleic acids are active in E. coli. E. coli cells that contain nucleic acids for both the upper MVA pathway and the lower MVA pathway as well as a kudzu isoprene synthase (strain MCM 127) produced significantly more isoprene (874 ng/L) compared to E. coli cells with nucleic acids for only the lower MVA pathway and the kudzu isoprene synthase (strain MCM 131) (see Table 3 and Example 8, part VIII).

In some embodiments, at least a portion of the cells maintain the heterologous isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acid for at least about 5, 10, 20, 50, 75, 100, 200, 300, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments of any of the aspects described herein, the nucleic acid comprising the heterologous or duplicate copy of an endogenous isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acid also comprises a selective marker, such as a kanamycin, ampicillin, carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol antibiotic resistance nucleic acid.

As indicated in Example 7, part VI, the amount of isoprene produced can be further increased by adding yeast extract to the cell culture medium. In this example, the amount of isoprene produced was linearly proportional to the amount of yeast extract in the cell medium for the concentrations tested (FIG. 48C). Additionally, approximately 0.11 grams of isoprene per liter of broth was produced from a cell medium with yeast extract and glucose (Example 7, part VIII). Both of these experiments used E. coli cells with kudzu isoprene synthase, S. cerevisia IDI, and E. coli DXS nucleic acids to produce isoprene. Increasing the amount of yeast extract in the presence of glucose resulted in more isoprene being produced than increasing the amount of glucose in the presence of yeast extract. Also, increasing the amount of yeast extract allowed the cells to produce a high level of isoprene for a longer length of time and improved the health of the cells.

Isoprene production was also demonstrated using three types of hydrolyzed biomass (bagasse, corn stover, and soft wood pulp) as the carbon source (FIGS. 46A-C). E. coli cells with kudzu isoprene synthase, S. cerevisia IDI, and E. coli DXS nucleic acids produced as much isoprene from these hydrolyzed biomass carbon sources as from the equivalent amount of glucose (e.g., 1% glucose, w/v). If desired, any other biomass carbon source can be used in the compositions and methods described herein. Biomass carbon sources are desirable because they are cheaper than many conventional cell mediums, thereby facilitating the economical production of isoprene.

Additionally, invert sugar was shown to function as a carbon source for the generation of isoprene (FIGS. 47C and 96-98). For example, 2.4 g/L of isoprene was produced from cells expressing MVA pathway polypeptides and a Kudzu isoprene synthase (Example 8, part XV). Glycerol was as also used as a carbon source for the generation of 2.2 mg/L of isoprene from cells expressing a Kudzu isoprene synthase (Example 8, part XIV). Expressing a DXS nucleic acid, an IDI nucleic acid, and/or one or more MVA pathway nucleic acids (such as nucleic acids encoding the entire MVA pathway) in addition to an isoprene synthase nucleic acid may increase the production of isoprene from glycerol.

In some embodiments, an oil is included in the cell medium. For example, B. subtilis cells containing a kudzu isoprene synthase nucleic acid produced isoprene when cultured in a cell medium containing an oil and a source of glucose (Example 4, part III). As another example, E. coli fadR atoC mutant cells containing the upper and lower MVA pathway plus kudzu isoprene synthase produced isoprene when cultured in a cell medium containing palm oil and a source of glucose (Example 27, part II). In some embodiments, more than one oil (such as 2, 3, 4, 5, or more oils) is included in the cell medium. While not intending to be bound to any particular theory, it is believed that (i) the oil may increase the amount of carbon in the cells that is available for conversion to isoprene, (ii) the oil may increase the amount of acetyl-CoA in the cells, thereby increasing the carbon flow through the MVA pathway, and/or (ii) the oil may provide extra nutrients to the cells, which is desirable since much of the carbon in the cells is converted to isoprene rather than other products. In some embodiments, cells that are cultured in a cell medium containing oil naturally use the MVA pathway to produce isoprene or are genetically modified to contain nucleic acids for the entire MVA pathway. In some embodiments, the oil is partially or completely hydrolyzed before being added to the cell culture medium to facilitate the use of the oil by the host cells.

One of the major hurdles to commercial production of small molecules such as isoprene in cells (e.g., bacteria) is the decoupling of production of the molecule from growth of the cells. In some embodiments for the commercially viable production of isoprene, a significant amount of the carbon from the feedstock is converted to isoprene, rather than to the growth and maintenance of the cells (“carbon efficiency”). In various embodiments, the cells convert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culture medium into isoprene. In particular embodiments, a significant portion of the carbon from the feedstock that is converted to downstream products is converted to isoprene. As described further in Example 11, E. coli cells expressing MVA pathway and kudzu isoprene synthase nucleic acids exhibited decoupling of the production of isoprene or the intermediate mevalonic acid from growth, resulting in high carbon efficiency. In particular, mevalonic acid was formed from cells expressing the upper MVA pathway from Enterococcus faecalis. Isoprene was formed from cells expressing the upper MVA pathway from Enterococcus faecalis, the lower MVA pathway from Saccharomyces cerevisiae, and the isoprene synthase from Pueraria montana (Kudzu). This decoupling of isoprene or mevalonic acid production from growth was demonstrated in four different strains of E. coli: BL21(LDE3), BL21(LDE3) Tuner, FM5, and MG1655. The first two E. coli strains are B strains, and the latter two are K12 strains. Decoupling of production from growth was also demonstrated in a variant of MG1655 with ack and pta genes deleted. This variant also demonstrated less production of acetate.

Exemplary Polypeptides and Nucleic Acids

Various isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptides and nucleic acids can be used in the compositions and methods described herein.

In some embodiments, the fusion polypeptide includes part or all of a first polypeptide (e.g., an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptide or catalytically active fragment thereof) and may optionally include part or all of a second polypeptide (e.g., a peptide that facilitates purification or detection of the fusion polypeptide, such as a His-tag). In some embodiments, the fusion polypeptide has an activity of two or more MVA pathway polypeptides (such as AA-CoA thiolase and HMG-CoA reductase polypeptides). In some embodiments, the polypeptide is a naturally-occurring polypeptide (such as the polypeptide encoded by an Enterococcus faecalis mvaE nucleic acid) that has an activity of two or more MVA pathway polypeptides.

In various embodiments, a polypeptide has at least or about 50, 100, 150, 175, 200, 250, 300, 350, 400, or more amino acids. In some embodiments, the polypeptide fragment contains at least or about 25, 50, 75, 100, 150, 200, 300, or more contiguous amino acids from a full-length polypeptide and has at least or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of an activity of a corresponding full-length polypeptide. In particular embodiments, the polypeptide includes a segment of or the entire amino acid sequence of any naturally-occurring isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptide. In some embodiments, the polypeptide has one or more mutations compared to the sequence of a wild-type (i.e., a sequence occurring in nature) isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptide.

In some embodiments, the polypeptide is an isolated polypeptide. In some embodiments, the polypeptide is a heterologous polypeptide.

In some embodiments, the nucleic acid is a recombinant nucleic acid. In some embodiments, an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid is operably linked to another nucleic acid encoding all or a portion of another polypeptide such that the recombinant nucleic acid encodes a fusion polypeptide that includes an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptide and all or part of another polypeptide (e.g., a peptide that facilitates purification or detection of the fusion polypeptide, such as a His-tag). In some embodiments, part or all of a recombinant nucleic acid is chemically synthesized. It is to be understood that mutations, including single nucleotide mutations, can occur within a nucleic acid as defined herein.

In some embodiments, the nucleic acid is a heterologous nucleic acid. In particular embodiments, the nucleic acid includes a segment of or the entire nucleic acid sequence of any naturally-occurring isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid. In some embodiments, the nucleic acid includes at least or about 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, or more contiguous nucleotides from a naturally-occurring isoprene synthase nucleic acid DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid. In some embodiments, the nucleic acid has one or more mutations compared to the sequence of a wild-type (i.e., a sequence occurring in nature) isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid. In some embodiments, the nucleic acid has one or more mutations (e.g., a silent mutation) that increase the transcription or translation of isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, or transcription factor nucleic acid. In some embodiments, the nucleic acid is a degenerate variant of any nucleic acid encoding an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptide.

The accession numbers of exemplary isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids are listed in Appendix 1 (the accession numbers of Appendix 1 and their corresponding sequences are herein incorporated by reference in their entireties, particularly with respect to the amino acid and nucleic acid sequences of isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids). The Kegg database also contains the amino acid and nucleic acid sequences of numerous exemplary isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids (see, for example, the world-wide web at “genome.jp/kegg/pathway/map/map00100.html” and the sequences therein, which are each hereby incorporated by reference in their entireties, particularly with respect to the amino acid and nucleic acid sequences of isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids). In some embodiments, one or more of the isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and/or nucleic acids have a sequence identical to a sequence publicly available on Dec. 12, 2007 or Sep. 14, 2008 such as any of the sequences that correspond to any of the accession numbers in Appendix 1 or any of the sequences present in the Kegg database. Additional exemplary isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids are described further below.

Exemplary Isoprene Synthase Polypeptides and Nucleic Acids

As noted above, isoprene synthase polypeptides convert dimethyl allyl diphosphate (DMAPP) into isoprene. Exemplary isoprene synthase polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of an isoprene synthase polypeptide. Standard methods can be used to determine whether a polypeptide has isoprene synthase polypeptide activity by measuring the ability of the polypeptide to convert DMAPP into isoprene in vitro, in a cell extract, or in vivo. In an exemplary assay, cell extracts are prepared by growing a strain (e.g., the E. coli/pTrcKudzu strain described herein) in the shake flask method as described in Example 1. After induction is complete, approximately 10 mL of cells are pelleted by centrifugation at 7000×g for 10 minutes and re-suspended in 5 ml of PEB without glycerol. The cells are lysed using a French Pressure cell using standard procedures. Alternatively the cells are treated with lysozyme (Ready-Lyse lysozyme solution; EpiCentre) after a freeze/thaw at −80° C.

Isoprene synthase polypeptide activity in the cell extract can be measured, for example, as described in Silver et al., J. Biol. Chem. 270:13010-13016, 1995 and references therein, which are each hereby incorporated by reference in their entireties, particularly with respect to assays for isoprene synthase polypeptide activity. DMAPP (Sigma) is evaporated to dryness under a stream of nitrogen and re-hydrated to a concentration of 100 mM in 100 mM potassium phosphate buffer pH 8.2 and stored at −20° C. To perform the assay, a solution of 5 μL of 1M MgCl₂, 1 mM (250 μg/ml) DMAPP, 65 μL of Plant Extract Buffer (PEB) (50 mM Tris-HCl, pH 8.0, 20 mM MgCl₂, 5% glycerol, and 2 mM DTT) is added to 25 μL of cell extract in a 20 ml Headspace vial with a metal screw cap and teflon coated silicon septum (Agilent Technologies) and cultured at 37° C. for 15 minutes with shaking. The reaction is quenched by adding 200 μL of 250 mM EDTA and quantified by GC/MS as described in Example 1, part II.

Exemplary isoprene synthase nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of an isoprene synthase polypeptide. Exemplary isoprene synthase polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.

In some embodiments, the isoprene synthase polypeptide or nucleic acid is from the family Fabaceae, such as the Faboideae subfamily. In some embodiments, the isoprene synthase polypeptide or nucleic acid is a polypeptide or nucleic acid from Pueraria montana (kudzu) (Sharkey et al., Plant Physiology 137: 700-712, 2005), Pueraria lobata, poplar (such as Populus alba, Populus nigra, Populus trichocarpa, or Populus alba x tremula (CAC35696) Miller et al., Planta 213: 483-487, 2001) aspen (such as Populus tremuloides) Silver et al., JBC 270(22): 13010-1316, 1995), or English Oak (Quercus robur) (Zimmer et al., WO 98/02550), which are each hereby incorporated by reference in their entireties, particularly with respect to isoprene synthase nucleic acids and the expression of isoprene synthase polypeptides. Suitable isoprene synthases include, but are not limited to, those identified by Genbank Accession Nos. AY341431, AY316691, AY279379, AJ457070, and AY182241, which are each hereby incorporated by reference in their entireties, particularly with respect to sequences of isoprene synthase nucleic acids and polypeptides. In some embodiments, the isoprene synthase polypeptide or nucleic acid is not a naturally-occurring polypeptide or nucleic acid from Quercus robur (i.e., the isoprene synthase polypeptide or nucleic acid is an isoprene synthase polypeptide or nucleic acid other than a naturally-occurring polypeptide or nucleic acid from Quercus robur). In some embodiments, the isoprene synthase nucleic acid or polypeptide is a naturally-occurring polypeptide or nucleic acid from poplar. In some embodiments, the isoprene synthase nucleic acid or polypeptide is not a naturally-occurring polypeptide or nucleic acid from poplar.

Exemplary DXS Polypeptides and Nucleic Acids

As noted above, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) polypeptides convert pyruvate and D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate. Exemplary DXS polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of a DXS polypeptide. Standard methods (such as those described herein) can be used to determine whether a polypeptide has DXS polypeptide activity by measuring the ability of the polypeptide to convert pyruvate and D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate in vitro, in a cell extract, or in vivo. Exemplary DXS nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of a DXS polypeptide. Exemplary DXS polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.

Exemplary IDI Polypeptides and Nucleic Acids

Isopentenyl diphosphate isomerase polypeptides (isopentenyl-diphosphate delta-isomerase or IDI) catalyses the interconversion of isopentenyl diphosphate (IPP) and dimethyl allyl diphosphate (DMAPP) (e.g., converting IPP into DMAPP and/or converting DMAPP into IPP). Exemplary IDI polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of an IDI polypeptide. Standard methods (such as those described herein) can be used to determine whether a polypeptide has IDI polypeptide activity by measuring the ability of the polypeptide to interconvert IPP and DMAPP in vitro, in a cell extract, or in vivo. Exemplary IDI nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of an IDI polypeptide. Exemplary IDI polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.

Exemplary MVA Pathway Polypeptides and Nucleic Acids

Exemplary MVA pathway polypeptides include acetyl-CoA acetyltransferase (AA-CoA thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK) polypeptides, phosphomevalonate kinase (PMK) polypeptides, diphosphomevalonate decarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC) polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDI polypeptides, and polypeptides (e.g., fusion polypeptides) having an activity of two or more MVA pathway polypeptides. In particular, MVA pathway polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of an MVA pathway polypeptide. Exemplary MVA pathway nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of an MVA pathway polypeptide. Exemplary MVA pathway polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.

In particular, acetyl-CoA acetyltransferase polypeptides (AA-CoA thiolase or AACT) convert two molecules of acetyl-CoA into acetoacetyl-CoA. Standard methods (such as those described herein) can be used to determine whether a polypeptide has AA-CoA thiolase polypeptide activity by measuring the ability of the polypeptide to convert two molecules of acetyl-CoA into acetoacetyl-CoA in vitro, in a cell extract, or in vivo.

3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase or HMGS) polypeptides convert acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA. Standard methods (such as those described herein) can be used to determine whether a polypeptide has HMG-CoA synthase polypeptide activity by measuring the ability of the polypeptide to convert acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA in vitro, in a cell extract, or in vivo.

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase or HMGR) polypeptides convert 3-hydroxy-3-methylglutaryl-CoA into mevalonate. Standard methods (such as those described herein) can be used to determine whether a polypeptide has HMG-CoA reductase polypeptide activity by measuring the ability of the polypeptide to convert 3-hydroxy-3-methylglutaryl-CoA into mevalonate in vitro, in a cell extract, or in vivo.

Mevalonate kinase (MVK) polypeptides phosphorylates mevalonate to form mevalonate-5-phosphate. Standard methods (such as those described herein) can be used to determine whether a polypeptide has MVK polypeptide activity by measuring the ability of the polypeptide to convert mevalonate into mevalonate-5-phosphate in vitro, in a cell extract, or in vivo.

Phosphomevalonate kinase (PMK) polypeptides phosphorylates mevalonate-5-phosphate to form mevalonate-5-diphosphate. Standard methods (such as those described herein) can be used to determine whether a polypeptide has PMK polypeptide activity by measuring the ability of the polypeptide to convert mevalonate-5-phosphate into mevalonate-5-diphosphate in vitro, in a cell extract, or in vivo.

Diphosphomevalonate decarboxylase (MVD or DPMDC) polypeptides convert mevalonate-5-diphosphate into isopentenyl diphosphate (IPP). Standard methods (such as those described herein) can be used to determine whether a polypeptide has MVD polypeptide activity by measuring the ability of the polypeptide to convert mevalonate-5-diphosphate into IPP in vitro, in a cell extract, or in vivo.

Phosphomevalonate decarboxylase (PMDC) polypeptides convert mevalonate-5-phosphate into isopentenyl phosphate (IP). Standard methods (such as those described herein) can be used to determine whether a polypeptide has PMDC polypeptide activity by measuring the ability of the polypeptide to convert mevalonate-5-phosphate into IP in vitro, in a cell extract, or in vivo.

Isopentenyl phosphate kinase (IPK) polypeptides phosphorylate isopentyl phosphate (IP) to form isopentenyl diphosphate (IPP). Standard methods (such as those described herein) can be used to determine whether a polypeptide has IPK polypeptide activity by measuring the ability of the polypeptide to convert IP into IPP in vitro, in a cell extract, or in vivo.

Exemplary IDI polypeptides and nucleic acids are described above.

Exemplary Hydrogenase Polypeptides and Nucleic Acids

Hydrogenase polypeptides catalyze the reaction: 2H⁺+2e⁻

H₂. In vitro that reaction is reversible, but certain hydrogenases may work in only one direction in vivo, either oxidizing H₂ or reducing H⁺. Hydrogenase polypeptides can be oxygen-sensitive, contain complex metal cofactors as part of their catalytic center and sometimes consist of multiple subunits, with hydrogenase gene expression sometimes involving additional accessory polypeptides, such as ‘maturation’ factors or transcription regulatory factors (i.e., activators or repressors). Hydrogenases are classified into at least three broad groups based upon the type of metal cofactor in their catalytic center: (1) nickel-iron (“NiFe”) hydrogenases have a nickel/iron cofactor; (2) iron-iron hydrogenases (“FeFe”) have an iron/iron cofactor; and (3) iron/sulfur-free (“Fe”) hydrogenases, which lack the 4Fe4S clusters found in groups (1) and (2), have an iron cofactor and a methenyl-tetrahydromethanopterin electron carrier. See, e.g., Chung-Jung Chou et al., “Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels,” Metabol. Eng. 10:394-404 (2008), and Gon111 Vardar-Schara et al., “Metabolically engineered bacteria for producing hydrogen via fermentation,” Microbial Biotechnol. 1(2):107-125 (2008), both of which are incorporated herein by reference in their entireties, particularly with respect to the various types and classes of hydrogenases. Although many organisms contain multiple hydrogenases, few contain genes for both NiFe and FeFe hydrogenases.

The catalytic center of NiFe hydrogenases consists of a nickel atom and an iron atom, each with two carbon monoxide (CO) and two cyanide (CN⁻) ligands. The NiFe hydrogenases all comprise at least a second subunit containing multiple iron-sulfur (Fe—S) centers for the transfer of electrons to and from the catalytic center. The NiFe hydrogenases can be subdivided into four main classes: (1) respiratory enzymes, which are part of multi-enzyme systems that couple the oxidation of H₂ to reduction of terminal electron acceptors such as SO₄ ²⁻ or NO₃ ⁻ under anaerobic conditions, or to O₂ in aerobic microorganisms; (2) H₂ sensors, which activate expression of the metabolically active NiFe hydrogenases; (3) cytoplasmic hydrogenases, containing multiple subunits able to utilize NADP⁺, which are readily reversible in vitro, but in vivo may only oxidize H₂; and (4) membrane-bound, energy-conserving multi-enzyme complexes also found in bacteria and Archaea. Chung-Jung Chou et al., “Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels,” Metabol. Eng. 10:394-404 (2008).

The catalytic center of FeFe hydrogenases contains a catalytic “H cluster” which coordinates a binuclear (FeFe) site bridged to a [4Fe-4S] center by a single protein (cysteine) ligand. The two iron atoms of the binuclear center each have two carbon monoxide (CO) and two cyanide (CN⁻s) ligands, and are also bridged by two sulfur atoms which are part of a small organic molecule. Most FeFe hydrogenases are monomeric enzymes of about 50 kilodaltons (kDa), and appear to function in vivo primarily to dispose of excess reducing equivalents by reducing protons to hydrogen gas. Chung-Jung Chou et al., “Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels,” Metabol. Eng. 10:394-404 (2008).

The catalytic center of Fe hydrogenases was originally thought to have an active site based on an organic cofactor with no metals involved, but was later shown to contain a mononuclear Fe atom. Despite the phylogenetic differences between the three types of hydrogenase, in addition to at least one iron atom, all three groups of hydrogenases also contain at least one carbon monoxide (CO) ligand to the iron atom in their active sites, which facilitates the catalytic oxidation of H₂ and the reduction of protons. Chung-Jung Chou et al., “Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels,” Metabol. Eng. 10:394-404 (2008).

Exemplary hydrogenase polypeptides include, but are not limited to, the E. coli hydrogenase-1 (Hyd-1) polypeptides, E. coli hydrogenase-2 (Hyd-2) polypeptides, E. coli hydrogenase-3 (Hyd-3) polypeptides, E. coli hydrogenase-4 (Hyd-4) polypeptides, E. coli formate hydrogen lyase (FHL) complex, which produces hydrogen gas from formate and CO₂ under anaerobic conditions at acidic pH (see, e.g., Akihito Yoshida et al., “Efficient induction of formate hydrogen lyase of aerobically grown Escherichia coli in a three-step biohydrogen production process,” Appl. Microbiol. Biotechnol. 74:754-760 (2007), which is incorporated herein by reference in its entirety, particularly with respect to the induction of expression of formate hydrogen lyase in E. coli), Ralstonia eutropha H16 hydrogenase (R. eutropha HoxH) Rhodococcus opacus MR11 hydrogenase (R. opacus HoxH) polypeptides, Synechosystis sp. PCC 6803 hydrogenase (Syn. PCC 6803 HoxH) polypeptides, Desulfovibrio gigas hydrogenase (D. gigas) polypeptides, and Desulfovibrio desulfuricans ATCC 7757 hydrogenase (D. desulfuricans) polypeptides (see, e.g., Gönül Vardar-Schara et al., “Metabolically engineered bacteria for producing hydrogen via fermentation,” Microbial Biotechnol. 1(2):107-125 (2008), which is incorporated herein by reference in its entirety, particularly with respect to the various types and classes of hydrogenases) and polypeptides (e.g., fusion polypeptides) having an activity of two or more hydrogenase polypeptides. In particular, hydrogenase polypeptides include polypeptides, fragments of polypeptides, peptides, and fusion polypeptides that have at least one activity of a hydrogenase polypeptide. Exemplary hydrogenase nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of a hydrogenase polypeptide, or at least one activity necessary for expression, processing, or maturation of a hydrogenase polypeptide. Exemplary hydrogenase polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.

E. coli Hyd-3, which is part of the anaerobic formate hydrogen lyase (FHL) complex, is encoded by the hyc operon (comprising the hycA, hycB, hycC, hycD, hycE, hycF, hycG, hycH, and hycI genes). E. coli Hyd-4 is encoded by the hyf operon (comprising the hyfA, hyfB, hyfC, hyfD, hyfE, hyfF, hyfG, hyfH, hyfI, hyfJ, and hyfR genes). E. coli FHL is encoded by six genes from the hyc operon (hycB, hycC, hycD, hycE, hycF and hycG) and the fdhF gene (encoding formate dehydrogenase H (Fdh-H)). Expression of the FHL complex can further involve expression of pyruvate formate lyase (pfl), FhlA, a transcription factor that activates transcription of fdhF and the hyc operon, or deletion/inactivation of HycA, a transcription factor encoded by the hycA gene that negatively regulates transcription of FHL. Co-production of isoprene and hydrogen can be improved by expression or inactivation/deletion of additional proteins involved in the regulation of gene expression for hydrogenases and other enzymes, such as, for example, iron-sulfur complex transcriptional regulator (iscR) (Kalim-Akhtar et al., “Deletion of iscR stimulates recombinant Clostridial Fe/Fe hydrogenase activity and H₂-accumulation in Escherichia coli BL21(DE3),” Appl. Microbiol. Biotechnol. 78:853-862 (2008), which is incorporated herein by reference in its entirety, particularly with reference to stimulation of Clostridial Fe/Fe hydrogenase activity and hydrogen accumulation in E. coli by deleting the iscR gene).

Exemplary ferredoxin-dependent hydrogenase polypeptides include, but are not limited to, Clostridium acetobutulicum hydrogenase A (HydA) (see, e.g., P. W. King et al., “Functional studies of [FeFe] hydrogenase maturation in an Escherichia coli biosynthetic system,” J. Bacteriol. 188(6):163-172 (2006), which is incorporated herein by reference in its entirety, particularly with respect to production of hydrogen by HydA and three HydA-associated maturation enzymes (HydE, HydG, and HydF), which may be expressed alone or in conjunction with one or more of: (1) Bacillus subtilis NADPH ferredoxin oxidoreductase (NFOR) (see, e.g., Viet et al., (2008)), which is incorporated herein by reference in its entirety, particularly with respect to production of hydrogen by NFOR; see also PCT Publication No. WO/2007/089901, which is incorporated herein by reference in its entirety, particularly with respect to optimization of E. coli strains for production of hydrogen), Clostridium kluyveri NADH ferredoxin oxidoreductase (RnfCDGEAB) (Henning Seedorf et al., “The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features,” Proc. Nat'l Acad. Sci. U.S.A. 105(6):2128-2133 (2008), which is incorporated herein by reference in its entirety, particular with reference to NADH ferredoxin oxidoreductase, and with reference to components of the anaerobic ethanol-acetate fermentation pathway), or Clostridium pasteuranium ferredoxin oxidoreductase (Fdx); (2) glyceraldehyde-6-phosphate ferredoxin oxidoreductase (“GAPOR”); or (3) pyruvate ferredoxin oxidoreductase (“POR”), and polypeptides (e.g., fusion polypeptides) having an activity of two or more hydrogenase polypeptides or of one or more hydrogenase polypeptides and an activity of one or more ferredoxin-dependent oxidoreductases. In particular, ferredoxin-dependent hydrogenase polypeptides include polypeptides, fragments of polypeptides, peptides, and fusion polypeptides that have at least one activity of a ferredoxin-dependent hydrogenase polypeptide.

Exemplary NADPH-dependent hydrogenase polypeptides include, but are not limited to thermophilic hydrogenase polypeptides such as Pyrococcus furiosus hydrogenase (see, e.g., J. Woodward et al., “Enzymatic production of biohydrogen,” Nature 405(6790):1014-1015 (2000)), and polypeptides (e.g., fusion polypeptides) having an activity of two or more NADPH-dependent hydrogenase polypeptides. In particular, NADPH-dependent hydrogenase polypeptides include polypeptides, fragments of polypeptides, peptides, and fusion polypeptides that have at least one activity of a NADPH-dependent hydrogenase polypeptide.

Exemplary oxygen-tolerant or oxygen-insensitive hydrogenases include, but are not limited to, Rubrivivax gelatinosus hydrogenase (see, e.g., P. C. Maness et al., “Characterization of the oxygen tolerance of a hydrogenase linked to a carbon monoxide oxidation pathway in Rubrivivax gelatinosus,” Appl. Environ. Microbiol. 68(6):2633-2636 (2002), which is incorporated herein by reference in its entirety, particularly with respect to R. gelatinosus hydrogenase), and Ralstonia eutropha hydrogenase polypeptides (see, e.g., T. Burgdorf et al., “[NiFe]-hydrogenases of Ralstonia eutropha H16: modular enzymes for oxygen-tolerant biological hydrogen oxidation,” J. Mol. Microbiol. Biotechnol. 10(2-4):181-196 (2005), which is incorporated herein by reference in its entirety, particularly with respect to R. eutropha hydrogenase polypeptides). Alternatively, heterologous nucleic acids encoding hydrogenase polypeptides can be mutagenized and screened for O₂-tolerance or O₂-insensitivity using standard methods and assays (see, e.g., L. E. Nagy et al., “Application of gene-shuffling for the rapid generation of novel [FeFe]-hydrogenase libraries,” Biotechnol. Letts. 29(3)421-430 (2007), which is incorporated herein by reference, particularly with respect to mutagenesis and screening for oxygen tolerant hydrogenase polypeptides).

Standard methods (such as those described herein) can be used to determine whether a polypeptide has hydrogenase activity by measuring the ability of the polypeptide to produce hydrogen gas in vitro, in a cell extract, or in vivo.

Exemplary Polypeptides and Nucleic Acids for Genes Related to Production of Fermentation Side Products

In addition to expressing or over-expressing heterologous or native hydrogenases in E. coli, co-production of isoprene and hydrogen can be improved by inactivation of anaerobic biosynthetic pathways, thereby blocking the carbon flow to a variety of metabolites (i.e., fermentation side products) produced under oxygen-limited or anaerobic conditions, including, but not limited to, lactate, acetate, pyruvate, ethanol, succinate, and glycerol. Exemplary polypeptides involved in the production of fermentation side products include formate dehydrogenase N, alpha subunit (fdnG), formate dehydrogenase O, large subunit (fdoG), nitrate reductase (narG), formate transporter A (focA), formate transporter B (focB), pyruvate oxidase (poxB), pyruvate dehydrogenase E1 component ackA/pta (aceE), alcohol dehydrogenase (adhE), fumarate reductase membrane protein (frdC), and lactate dehydrogenase (ldhA). See, e.g., Toshinori Maeda et al., “Enhanced hydrogen production from glucose by metabolically engineered Escherichia coli,” Appl. Microbiol. Biotechnol. 77(4):879-890 (2007), which is incorporated by reference in its entirety, particularly with respect to production of E. coli strains with modified glucose metabolism. Exemplary polypeptides involved in the regulation or expression of genes involved in the production of fermentation side products that may also be inactivated to improve co-production of isoprene and hydrogen include, but are not limited to, repressor of formate hydrogen lyase (hycA), fumarate reductase regulator (fnr), acetyl-coenzyme A synthetase (acs), and formate dehydrogenase regulatory protein (hycA), which regulates expression of the transcriptional regulator fhlA (formate hydrogen lyase transcriptional activator).

Exemplary Polypeptides and Nucleic Acids for Genes Related to Hydrogen Re-Uptake

Exemplary polypeptides involved in hydrogen re-uptake that may also be inactivated to improve co-production of isoprene and hydrogen include, but are not limited to, E. coli hydrogenase-1 (Hyd-1) (hya operon) and E. coli hydrogenase-2 (Hyd-2) (hyb operon). E. coli Hyd-1 is encoded by the hya operon (comprising the hyaA, hyaB, hyaC, hyaD, hyaE, and hyaF genes). E. coli Hyd-2 is encoded by the hyb operon (comprising the hybA, hybB, hybC, hybD, hybE, hybF, hybG, and hybO genes).

Exemplary Methods for Isolating Nucleic Acids

Isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids can be isolated using standard methods. Methods of obtaining desired nucleic acids from a source organism of interest (such as a bacterial genome) are common and well known in the art of molecular biology (see, for example, WO 2004/033646 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to the isolation of nucleic acids of interest). For example, if the sequence of the nucleic acid is known (such as any of the known nucleic acids described herein), suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired nucleic acid sequence. Once the sequence is isolated, the DNA may be amplified using standard primer directed amplification methods such as polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202, which is incorporated by reference in its entirety, particularly with respect to PCR methods) to obtain amounts of DNA suitable for transformation using appropriate vectors.

Alternatively, isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids (such as any isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids with a known nucleic acid sequence) can be chemically synthesized using standard methods.

Additional isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptides and nucleic acids which may be suitable for use in the compositions and methods described herein can be identified using standard methods. For example, cosmid libraries of the chromosomal DNA of organisms known to produce isoprene naturally can be constructed in organisms such as E. coli, and then screened for isoprene production. In particular, cosmid libraries may be created where large segments of genomic DNA (35-45 kb) are packaged into vectors and used to transform appropriate hosts. Cosmid vectors are unique in being able to accommodate large quantities of DNA. Generally cosmid vectors have at least one copy of the cos DNA sequence which is needed for packaging and subsequent circularization of the heterologous DNA. In addition to the cos sequence, these vectors also contain an origin of replication such as ColEI and drug resistance markers such as a nucleic acid resistant to ampicillin or neomycin. Methods of using cosmid vectors for the transformation of suitable bacterial hosts are well described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to transformation methods.

Typically to clone cosmids, heterologous DNA is isolated using the appropriate restriction endonucleases and ligated adjacent to the cos region of the cosmid vector using the appropriate ligases. Cosmid vectors containing the linearized heterologous DNA are then reacted with a DNA packaging vehicle such as bacteriophage. During the packaging process, the cos sites are cleaved and the heterologous DNA is packaged into the head portion of the bacterial viral particle. These particles are then used to transfect suitable host cells such as E. coli. Once injected into the cell, the heterologous DNA circularizes under the influence of the cos sticky ends. In this manner, large segments of heterologous DNA can be introduced and expressed in host cells.

Additional methods for obtaining isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids include screening a metagenomic library by assay (such as the headspace assay described herein) or by PCR using primers directed against nucleotides encoding for a length of conserved amino acids (for example, at least 3 conserved amino acids). Conserved amino acids can be identified by aligning amino acid sequences of known isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptides. Conserved amino acids for isoprene synthase polypeptides can be identified based on aligned sequences of known isoprene synthase polypeptides. An organism found to produce isoprene naturally can be subjected to standard protein purification methods (which are well known in the art) and the resulting purified polypeptide can be sequenced using standard methods. Other methods are found in the literature (see, for example, Julsing et al., Applied. Microbiol. Biotechnol. 75: 1377-84, 2007; Withers et al., Appl Environ Microbiol. 73(19):6277-83, 2007, which are each hereby incorporated by reference in their entireties, particularly with respect to identification of nucleic acids involved in the synthesis of isoprene).

Additionally, standard sequence alignment and/or structure prediction programs can be used to identify additional DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptides and nucleic acids based on the similarity of their primary and/or predicted polypeptide secondary structure with that of known DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptides and nucleic acids. Standard databases such as the swissprot-trembl database (world-wide web at “expasy.org”, Swiss Institute of Bioinformatics Swiss-Prot group CMU-1 rue Michel Servet CH-1211 Geneva 4, Switzerland) can also be used to identify isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription regulatory polypeptides and nucleic acids. The secondary and/or tertiary structure of an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptide can be predicted using the default settings of standard structure prediction programs, such as PredictProtein (630 West, 168 Street, BB217, New York, N.Y. 10032, USA). Alternatively, the actual secondary and/or tertiary structure of an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptide can be determined using standard methods. Additional isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids can also be identified by hybridization to probes generated from known isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids.

Exemplary Promoters and Vectors

Any of the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids described herein can be included in one or more vectors. Accordingly, also described herein are vectors with one more nucleic acids encoding any of the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptides that are described herein. In some embodiments, the vector contains a nucleic acid under the control of an expression control sequence.

In some embodiments, the vector contains a selective marker or selectable marker. Markers useful in vector systems for transformation of Trichoderma are known in the art (see, e.g., Finkelstein, Chapter 6 in Biotechnology of Filamentous Fungi, Finkelstein et al., Eds. Butterworth-Heinemann, Boston, Mass., Chap. 6., 1992; and Kinghorn et al., Applied Molecular Genetics of Filamentous Fungi, Blackie Academic and Professional, Chapman and Hall, London, 1992, which are each hereby incorporated by reference in their entireties, particularly with respect to selective markers). In some embodiments, the selective marker is the amdS nucleic acid, which encodes the enzyme acetamidase, allowing transformed cells to grow on acetamide as a nitrogen source. The use of an A. nidulans amdS nucleic acid as a selective marker is described in Kelley et al., EMBO J. 4:475-479, 1985 and Penttila et al., Gene 61:155-164, 1987 (which are each hereby incorporated by reference in their entireties, particularly with respect to selective markers). In some embodiments, an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation, or transcription regulatory nucleic acid integrates into a chromosome of the cells without a selective marker.

Suitable vectors are those which are compatible with the host cell employed. Suitable vectors can be derived, for example, from a bacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, a yeast, or a plant. Protocols for obtaining and using such vectors are known to those in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to the use of vectors).

Promoters are well known in the art. Any promoter that functions in the host cell can be used for expression of an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid in the host cell. Initiation control regions or promoters, which are useful to drive expression of isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids in various host cells are numerous and familiar to those skilled in the art (see, for example, WO 2004/033646 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to vectors for the expression of nucleic acids of interest). Virtually any promoter capable of driving these nucleic acids can be used including, but not limited to, CYC1, HIS3, GALL, GAL10, ADH1, PGK, PHO5, GAPDH, ADCI, TRP1, URA3, LEU2, ENO, and TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, trp, λP_(L), λP_(R), T7, tac, and trc (useful for expression in E. coli).

In some embodiments, a glucose isomerase promoter is used (see, for example, U.S. Pat. No. 7,132,527 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect promoters and plasmid systems for expressing polypeptides of interest). Reported glucose isomerase promoter mutants can be used to vary the level of expression of the polypeptide encoded by a nucleic acid operably linked to the glucose isomerase promoter (U.S. Pat. No. 7,132,527). In various embodiments, the glucose isomerase promoter is contained in a low, medium, or high copy plasmid (U.S. Pat. No. 7,132,527).

In various embodiments, an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is contained in a low copy plasmid (e.g., a plasmid that is maintained at about 1 to about 4 copies per cell), medium copy plasmid (e.g., a plasmid that is maintained at about 10 to about 15 copies per cell), or high copy plasmid (e.g., a plasmid that is maintained at about 50 or more copies per cell). In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is operably linked to a T7 promoter. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid operably linked to a T7 promoter is contained in a medium or high copy plasmid. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is operably linked to a Trc promoter. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid operably linked to a Trc promoter is contained in a medium or high copy plasmid. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is operably linked to a Lac promoter. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid operably linked to a Lac promoter is contained in a low copy plasmid. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is operably linked to an endogenous promoter, such as an endogenous Escherichia, Panteoa, Bacillus, Yarrowia, Streptomyces, or Trichoderma promoter or an endogenous alkaline serine protease, isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor promoter. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid operably linked to an endogenous promoter is contained in a high copy plasmid. In some embodiments, the vector is a replicating plasmid that does not integrate into a chromosome in the cells. In some embodiments, part or all of the vector integrates into a chromosome in the cells.

In some embodiments, the vector is any vector which when introduced into a fungal host cell is integrated into the host cell genome and is replicated. Reference is made to the Fungal Genetics Stock Center Catalogue of Strains (FGSC, the world-wide web at “fgsc.net” and the references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to vectors) for a list of vectors. Additional examples of suitable expression and/or integration vectors are provided in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989, Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds) 1987, Supplement 30, section 7.7.18); van den Hondel et al. in Bennett and Lasure (Eds.) More Gene Manipulations in Fungi, Academic Press pp. 396-428, 1991; and U.S. Pat. No. 5,874,276, which are each hereby incorporated by reference in their entireties, particularly with respect to vectors. Particularly useful vectors include pFB6, pBR322, PUC18, pUC100, and pENTR/D.

In some embodiments, an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is operably linked to a suitable promoter that shows transcriptional activity in a fungal host cell. The promoter may be derived from one or more nucleic acids encoding a polypeptide that is either endogenous or heterologous to the host cell. In some embodiments, the promoter is useful in a Trichoderma host. Suitable non-limiting examples of promoters include cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2, xyn1, and amy. In some embodiments, the promoter is one that is native to the host cell. For example, in some embodiments when T. reesei is the host, the promoter is a native T. reesei promoter. In some embodiments, the promoter is T. reesei cbh1, which is an inducible promoter and has been deposited in GenBank under Accession No. D86235, which is incorporated by reference in its entirety, particularly with respect to promoters. In some embodiments, the promoter is one that is heterologous to the fungal host cell. Other examples of useful promoters include promoters from the genes of A. awamori and A. niger glucoamylase (glaA) (Nunberg et al., Mol. Cell. Biol. 4:2306-2315, 1984 and Boel et al., EMBO J. 3:1581-1585, 1984, which are each hereby incorporated by reference in their entireties, particularly with respect to promoters); Aspergillus niger alpha amylases, Aspergillus oryzae TAKA amylase, T. reesei xln1, and the T. reesei cellobiohydrolase 1 (EP 137280, which is incorporated by reference in its entirety, particularly with respect to promoters).

In some embodiments, the expression vector also includes a termination sequence. Termination control regions may also be derived from various genes native to the host cell. In some embodiments, the termination sequence and the promoter sequence are derived from the same source. In another embodiment, the termination sequence is endogenous to the host cell. A particularly suitable terminator sequence is cbh1 derived from a Trichoderma strain (such as T. reesei). Other useful fungal terminators include the terminator from an A. niger or A. awamori glucoamylase nucleic acid (Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boel et al., EMBO J. 3:1581-1585, 1984; which are each hereby incorporated by reference in their entireties, particularly with respect to fungal terminators). Optionally, a termination site may be included. For effective expression of the polypeptides, DNA encoding the polypeptide are linked operably through initiation codons to selected expression control regions such that expression results in the formation of the appropriate messenger RNA.

In some embodiments, the promoter, coding, region, and terminator all originate from the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid to be expressed. In some embodiments, the coding region for an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is inserted into a general-purpose expression vector such that it is under the transcriptional control of the expression construct promoter and terminator sequences. In some embodiments, genes or part thereof are inserted downstream of the strong cbh1 promoter.

An isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid can be incorporated into a vector, such as an expression vector, using standard techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is hereby incorporated by reference in its entirety, particularly with respect to the screening of appropriate DNA sequences and the construction of vectors). Methods used to ligate the DNA construct comprising a nucleic acid of interest (such as an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid), a promoter, a terminator, and other sequences and to insert them into a suitable vector are well known in the art. For example, restriction enzymes can be used to cleave the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid and the vector. Then, the compatible ends of the cleaved isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid and the cleaved vector can be ligated Linking is generally accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide linkers are used in accordance with conventional practice (see, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989, and Bennett and Lasure, More Gene Manipulations in Fungi, Academic Press, San Diego, pp 70-76, 1991, which are each hereby incorporated by reference in their entireties, particularly with respect to oligonucleotide linkers). Additionally, vectors can be constructed using known recombination techniques (e.g., Invitrogen Life Technologies, Gateway Technology).

In some embodiments, it may be desirable to over-express isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids at levels far higher than currently found in naturally-occurring cells. This result may be accomplished by the selective cloning of the nucleic acids encoding those polypeptides into multicopy plasmids or placing those nucleic acids under a strong inducible or constitutive promoter. Methods for over-expressing desired polypeptides are common and well known in the art of molecular biology and examples may be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to cloning techniques.

In some embodiments, it may be desirable to under-express (e.g., mutate, inactivate, or delete) isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation, or transcription factor polypeptide-encoding nucleic acids at levels far below that those currently found in naturally-occurring cells. This result may be accomplished by the mutation or inactivation of transcriptional regulatory proteins required for expression of isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids, by deletion of the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids, or by placing those nucleic acids under the control of a strong repressible promoter. Methods for mutating, inactivating, or deleting desired polypeptides are common and well known in the art of molecular biology and examples may be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to cloning and mutagenesis techniques.

The following resources include descriptions of additional general methodology useful in accordance with the compositions and methods described herein: Kreigler, Gene Transfer and Expression; A Laboratory Manual, 1990 and Ausubel et al., Eds. Current Protocols in Molecular Biology, 1994, which are each hereby incorporated by reference in their entireties, particularly with respect to molecular biology and cloning techniques.

Exemplary Source Organisms

Isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids (and their encoded polypeptides) can be obtained from any organism that naturally contains isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids. As noted above, isoprene is formed naturally by a variety of organisms, such as bacteria, yeast, plants, and animals. Organisms contain the MVA pathway, DXP pathway, or both the MVA and DXP pathways for producing isoprene (FIGS. 19A and 19B). Thus, DXS nucleic acids can be obtained, e.g., from any organism that contains the DXP pathway or contains both the MVA and DXP pathways. IDI and isoprene synthase nucleic acids can be obtained, e.g., from any organism that contains the MVA pathway, DXP pathway, or both the MVA and DXP pathways. MVA pathway nucleic acids can be obtained, e.g., from any organism that contains the MVA pathway or contains both the MVA and DXP pathways. Hydrogenase nucleic acids can be obtained, e.g., from any organism that oxidizes hydrogen or reduces hydrogen ions. Fermentation side product genes can be obtained or identified, e.g., from any organism that undergoes oxygen-limited or anaerobic respiration, such as glycolysis.

In some embodiments, the nucleic acid sequence of the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic is identical to the sequence of a nucleic acid that is produced by any of the following organisms in nature. In some embodiments, the amino acid sequence of the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptide is identical to the sequence of a polypeptide that is produced by any of the following organisms in nature. In some embodiments, the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid or polypeptide is a mutant nucleic acid or polypeptide derived from any of the organisms described herein. As used herein, “derived from” refers to the source of the nucleic acid or polypeptide into which one or more mutations is introduced. For example, a polypeptide that is “derived from a plant polypeptide” refers to polypeptide of interest that results from introducing one or more mutations into the sequence of a wild-type (i.e., a sequence occurring in nature) plant polypeptide.

In some embodiments, the source organism is a fungus, examples of which are species of Aspergillus such as A. oryzae and A. niger, species of Saccharomyces such as S. cerevisiae, species of Schizosaccharomyces such as S. pombe, and species of Trichoderma such as T. reesei. In some embodiments, the source organism is a filamentous fungal cell. The term “filamentous fungi” refers to all filamentous forms of the subdivision Eumycotina (see, Alexopoulos, C. J. (1962), Introductory Mycology, Wiley, New York). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism is obligatory aerobic. The filamentous fungal parent cell may be a cell of a species of, but not limited to, Trichoderma, (e.g., Trichoderma reesei, the asexual morph of Hypocrea jecorina, previously classified as T. longibrachiatum, Trichoderma viride, Trichoderma koningii, Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol. Biotechnol 20: 46-53, 1984; ATCC No. 56765 and ATCC No. 26921); Penicillium sp., Humicola sp. (e.g., H. insolens, H. lanuginose, or H. grisea); Chrysosporium sp. (e.g., C. lucknowense), Gliocladium sp., Aspergillus sp. (e.g., A. oryzae, A. niger, A. sojae, A. japonicus, A. nidulans, or A. awamori) (Ward et al., Appl. Microbiol. Biotechnol. 39: 7380743, 1993 and Goedegebuur et al., Genet. 41: 89-98, 2002), Fusarium sp., (e.g., F. roseum, F. graminum F. cerealis, F. oxysporuim, or F. venenatum), Neurospora sp., (e.g., N. crassa), Hypocrea sp., Mucor sp., (e.g., M. miehei), Rhizopus sp. and Emericella sp. (see also, Innis et al., Sci. 228: 21-26, 1985). The term “Trichoderma” or “Trichoderma sp.” or “Trichoderma spp.” refer to any fungal genus previously or currently classified as Trichoderma.

In some embodiments, the fungus is A. nidulans, A. awamori, A. oryzae, A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F. oxysporum, or F. solani. Aspergillus strains are disclosed in Ward et al., Appl. Microbiol. Biotechnol. 39:738-743, 1993 and Goedegebuur et al., Curr Gene 41:89-98, 2002, which are each hereby incorporated by reference in their entireties, particularly with respect to fungi. In particular embodiments, the fungus is a strain of Trichoderma, such as a strain of T. reesei. Strains of T. reesei are known and non-limiting examples include ATCC No. 13631, ATCC No. 26921, ATCC No. 56764, ATCC No. 56765, ATCC No. 56767, and NRRL 15709, which are each hereby incorporated by reference in their entireties, particularly with respect to strains of T. reesei. In some embodiments, the host strain is a derivative of RL-P37. RL-P37 is disclosed in Sheir-Neiss et al., Appl. Microbiol. Biotechnology 20:46-53, 1984, which is hereby incorporated by reference in its entirety, particularly with respect to strains of T. reesei.

In some embodiments, the source organism is a yeast, such as Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp. In some embodiments, the Saccharomyces sp. is Saccharomyces cerevisiae.

In some embodiments, the source organism is a bacterium, such as strains of Bacillus such as B. lichenformis or B. subtilis, strains of Pantoea such as P. citrea, strains of Pseudomonas such as P. alcaligenes, P. putida, or P. fluorescens, strains of Streptomyces such as S. lividans or S. rubiginosus, strains of Corynebacterium sp. such as Corynebacterium glutamicum, strains of Rhodopseudomonas sp. such as Rhodopseudomonas palustris, or strains of Escherichia such as E. coli.

As used herein, “the genus Bacillus” includes all species within the genus “Bacillus,” as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named “Geobacillus stearothermophilus.” The production of resistant endospores in the presence of oxygen is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

In some embodiments, the source organism is a gram-positive bacterium. Non-limiting examples include strains of Streptomyces (e.g., S. lividans, S. coelicolor, or S. griseus) and Bacillus. In some embodiments, the source organism is a gram-negative bacterium, such as E. coli., Rhodopseudomonas sp. such as Rhodopseudomonas palustris, or Pseudomonas sp., such as P. alcaligenes, P. putida, or P. fluorescens.

In some embodiments, the source organism is a plant, such as a plant from the family Fabaceae, such as the Faboideae subfamily. In some embodiments, the source organism is kudzu, poplar (such as Populus alba x tremula CAC35696), aspen (such as Populus tremuloides), or Quercus robur.

In some embodiments, the source organism is an algae, such as a green algae, red algae, glaucophytes, chlorarachniophytes, euglenids, chromista, or dinoflagellates.

In some embodiments, the source organism is a cyanobacteria, such as cyanobacteria classified into any of the following groups based on morphology: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales, or Stigonematales.

In some embodiments, the source organism is an anaerobic organism. Anaerobic organisms can include, but are not limited to, obligate anaerobes, facultatitive anaerobes, and aerotolerant anaerobes. Such organisms can be any of the organisms listed above, bacteria, yeast, etc. In one embodiment, the obligate anaerobes can be any one or combination selected from the group consisting of Clostridium ljungdahlii, Clostridium autoethanogenum, Eurobacterium limosum, Clostridium carboxydivorans, Peptostreptococcus productus, and Butyribacterium methylotrophicum. It is to be understood that any combination of any of the source organisms described herein can be used for other embodiments of the invention.

Exemplary Host Cells

A variety of host cells can be used to express isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptides and to co-produce isoprene and hydrogen in the methods described herein. Exemplary host cells include cells from any of the organisms listed in the prior section under the heading “Exemplary Source Organisms.” The host cell may be a cell that naturally produces isoprene or a cell that does not naturally produce isoprene. In some embodiments, the host cell naturally produces isoprene using the DXP pathway, and an isoprene synthase, DXS, and/or IDI nucleic acid is added to enhance production of isoprene using this pathway. In some embodiments, the host cell naturally produces isoprene using the MVA pathway, and an isoprene synthase and/or one or more MVA pathway nucleic acids are added to enhance production of isoprene using this pathway. In some embodiments, the host cell naturally produces isoprene using the DXP pathway and one or more MVA pathway nucleic acids are added to produce isoprene using part or all of the MVA pathway as well as the DXP pathway. In some embodiments, the host cell naturally produces isoprene using both the DXP and MVA pathways and one or more isoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added to enhance production of isoprene by one or both of these pathways.

In some embodiments, the host cell naturally produces isoprene using both the DXP and MVA pathways, and one or more isoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added to enhance production of isoprene by one or both of these pathways, one or more hydrogenase nucleic acids are added to enhance hydrogen production and one or more fermentation side product-producing genes are inactivated or deleted to limit production of fermentation side products. In some embodiments, the host cell naturally co-produces isoprene and hydrogen using both the DXP and MVA pathways and one or more isoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added to enhance production of isoprene by one or both of these pathways, one or more hydrogenase nucleic acids are added to enhance hydrogen production, one or more fermentation side product-producing genes are inactivated or deleted to limit production of fermentation side products, and one or more hydrogen reuptake genes are inactivated or deleted to increase hydrogen production. In some embodiments, the host cell naturally co-produces isoprene and hydrogen using both the DXP and MVA pathways and a hydrogenase, and one or more isoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added to enhance production of isoprene by one or both of these pathways, one or more hydrogenase nucleic acids are added to enhance hydrogen production, one or more hydrogenase maturation nucleic acids are added to enhance hydrogen production, one or more fermentation side product-producing genes are inactivated or deleted to limit production of fermentation side products, and one or more hydrogen reuptake genes are inactivated or deleted to increase hydrogen production. In some embodiments, the host cell naturally co-produces isoprene and hydrogen using both the DXP and MVA pathways and one or more isoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added to enhance production of isoprene by one or both of these pathways, one or more hydrogenase nucleic acids are added to enhance hydrogen production, one or more hydrogenase maturation nucleic acids are added to enhance hydrogen production, one or more transcription factor nucleic acids are added or inactivated or deleted to enhance hydrogenase production, one or more fermentation side product-producing genes are inactivated or deleted to limit production of fermentation side products, and one or more hydrogen reuptake genes are inactivated or deleted to increase hydrogen production.

Exemplary Transformation Methods

Isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids or vectors containing them can be inserted into a host cell (e.g., a plant cell, a fungal cell, a yeast cell, or a bacterial cell described herein) using standard techniques for expression of the encoded isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptide. Introduction of a DNA construct or vector into a host cell can be performed using techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated or DEAE-Dextrin mediated transfection or transfection using a recombinant phage virus), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, and protoplast fusion. General transformation techniques are known in the art (see, e.g., Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds) Chapter 9, 1987; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989; and Campbell et al., Curr. Genet. 16:53-56, 1989, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods). The expression of heterologous polypeptide in Trichoderma is described in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; U.S. Pat. No. 7,262,041; WO 2005/001036; Harkki et al., Enzyme Microb. Technol. 13:227-233, 1991; Harkki et al., Bio Technol. 7:596-603, 1989; EP 244,234; EP 215,594; and Nevalainen et al., “The Molecular Biology of Trichoderma and its Application to the Expression of Both Homologous and Heterologous Genes,” in Molecular Industrial Mycology, Eds. Leong and Berka, Marcel Dekker Inc., NY pp. 129-148, 1992, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation and expression methods). Reference is also made to Cao et al., (Sci. 9:991-1001, 2000; EP 238023; and Yelton et al., Proceedings. Natl. Acad. Sci. USA 81:1470-1474, 1984 (which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods) for transformation of Aspergillus strains. The introduced nucleic acids may be integrated into chromosomal DNA or maintained as extrachromosomal replicating sequences.

Any method known in the art may be used to select transformants. In one non-limiting example, stable transformants including an amdS marker are distinguished from unstable transformants by their faster growth rate and the formation of circular colonies with a smooth, rather than ragged outline on solid culture medium containing acetamide. Additionally, in some cases a further test of stability is conducted by growing the transformants on a solid non-selective medium (e.g., a medium that lacks acetamide), harvesting spores from this culture medium, and determining the percentage of these spores which subsequently germinate and grow on selective medium containing acetamide.

In some embodiments, fungal cells are transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a known manner. In one specific embodiment, the preparation of Trichoderma sp. for transformation involves the preparation of protoplasts from fungal mycelia (see, Campbell et al., Curr. Genet. 16:53-56, 1989, which is incorporated by reference in its entirety, particularly with respect to transformation methods). In some embodiments, the mycelia are obtained from germinated vegetative spores. The mycelia are treated with an enzyme that digests the cell wall resulting in protoplasts. The protoplasts are then protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Usually the concentration of these stabilizers varies between 0.8 M and 1.2 M. It is desirable to use about a 1.2 M solution of sorbitol in the suspension medium.

Uptake of DNA into the host Trichoderma sp. strain is dependent upon the calcium ion concentration. Generally, between about 10 mM CaCl₂ and 50 mM CaCl₂ is used in an uptake solution. In addition to the calcium ion in the uptake solution, other compounds generally included are a buffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethylene glycol (PEG). While not intending to be bound to any particular theory, it is believed that the polyethylene glycol acts to fuse the cell membranes, thus permitting the contents of the medium to be delivered into the cytoplasm of the Trichoderma sp. strain and the plasmid DNA to be transferred to the nucleus. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.

Usually a suspension containing the Trichoderma sp. protoplasts or cells that have been subjected to a permeability treatment at a density of 10⁵ to 10⁷/mL (such as 2×10⁶/mL) are used in the transformation. A volume of 100 μL of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaCl₂) are mixed with the desired DNA. Generally, a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension. In some embodiments, about 0.25 volumes are added to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, and the like may also be added to the uptake solution and aid in transformation. Similar procedures are available for other fungal host cells (see, e.g., U.S. Pat. Nos. 6,022,725 and 6,268,328, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods).

Generally, the mixture is then cultured at approximately 0° C. for a period of between 10 to 30 minutes. Additional PEG is then added to the mixture to further enhance the uptake of the desired nucleic acid sequence. The 25% PEG 4000 is generally added in volumes of 5 to 15 times the volume of the transformation mixture; however, greater and lesser volumes may be suitable. The 25% PEG 4000 is desirably about 10 times the volume of the transformation mixture. After the PEG is added, the transformation mixture is then cultured either at room temperature or on ice before the addition of a sorbitol and CaCl₂ solution. The protoplast suspension is then further added to molten aliquots of a growth medium. When the growth medium includes a growth selection (e.g., acetamide or an antibiotic) it permits the growth of transformants only.

The transformation of bacterial cells may be performed according to conventional methods, e.g., as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is hereby incorporated by reference in its entirety, particularly with respect to transformation methods.

Exemplary Cell Culture Media

Also described herein is a cell or a population of cells in culture that co-produce isoprene and hydrogen. By “cells in culture” is meant two or more cells in a solution (e.g., a cell growth medium) that allows the cells to undergo one or more cell divisions. “Cells in culture” do not include plant cells that are part of a living, multicellular plant containing cells that have differentiated into plant tissues. In various embodiments, the cell culture includes at least or about 10, 20, 50, 100, 200, 500, 1,000, 5,000, 10,000 or more cells.

By “cells in oxygen-limited culture” is meant two or more cells in a solution (e.g., a cell growth medium) that allows the cell to under go one or more cell divisions, wherein the solution contains a limiting amount of oxygen. The term “oxygen-limited culture” means that the culture is either anoxic or contains less than the required amount of oxygen to support respiration via the biological transfer of reducing equivalents to oxygen, and also encompasses anaerobic cultures. Under oxygen-limited culture conditions, some electrons derived from carbon metabolism cannot be accepted because oxygen concentrations are too low, causing cells to switch to hydrogen production if they comprise the appropriate metabolic pathways for doing so. Oxygen-limited culture conditions occur when the oxygen transfer rate (“OTR”) is less than the oxygen uptake rate (“OUR”) indicated by dissolved oxygen concentrations of close to zero in culture medium.

Any carbon source can be used to cultivate the host cells. The term “carbon source” refers to one or more carbon-containing compounds capable of being metabolized by a host cell or organism. For example, the cell medium used to cultivate the host cells may include any carbon source suitable for maintaining the viability or growing the host cells.

In some embodiments, the carbon source is a carbohydrate (such as monosaccharide, disaccharide, oligosaccharide, or polysaccharides), invert sugar (e.g., enzymatically treated sucrose syrup), glycerol, glycerine (e.g., a glycerine byproduct of a biodiesel or soap-making process), dihydroxyacetone, one-carbon source, oil (e.g., a plant or vegetable oil such as corn, palm, or soybean oil), animal fat, animal oil, fatty acid (e.g., a saturated fatty acid, unsaturated fatty acid, or polyunsaturated fatty acid), lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, polypeptide (e.g., a microbial or plant protein or peptide), renewable carbon source (e.g., a biomass carbon source such as a hydrolyzed biomass carbon source), yeast extract, component from a yeast extract, polymer, acid, alcohol, aldehyde, ketone, amino acid, succinate, lactate, acetate, ethanol, or any combination of two or more of the foregoing. In some embodiments, the carbon source is a product of photosynthesis, including, but not limited to, glucose.

Exemplary monosaccharides include glucose and fructose; exemplary oligosaccharides include lactose and sucrose, and exemplary polysaccharides include starch and cellulose. Exemplary carbohydrates include C6 sugars (e.g., fructose, mannose, galactose, or glucose) and C5 sugars (e.g., xylose or arabinose). In some embodiments, the cell medium includes a carbohydrate as well as a carbon source other than a carbohydrate (e.g., glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, or a component from a yeast extract). In some embodiments, the cell medium includes a carbohydrate as well as a polypeptide (e.g., a microbial or plant protein or peptide). In some embodiments, the microbial polypeptide is a polypeptide from yeast or bacteria. In some embodiments, the plant polypeptide is a polypeptide from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.

In some embodiments, the concentration of the carbohydrate is at least or about 5 grams per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such as at least or about 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, 400, or more g/L. In some embodiments, the concentration of the carbohydrate is between about 50 and about 400 g/L, such as between about 100 and about 360 g/L, between about 120 and about 360 g/L, or between about 200 and about 300 g/L. In some embodiments, this concentration of carbohydrate includes the total amount of carbohydrate that is added before and/or during the culturing of the host cells.

In some embodiments, the cells are cultured under limited glucose conditions. By “limited glucose conditions” is meant that the amount of glucose that is added is less than or about 105% (such as about 100%) of the amount of glucose that is consumed by the cells. In particular embodiments, the amount of glucose that is added to the culture medium is approximately the same as the amount of glucose that is consumed by the cells during a specific period of time. In some embodiments, the rate of cell growth is controlled by limiting the amount of added glucose such that the cells grow at the rate that can be supported by the amount of glucose in the cell medium. In some embodiments, glucose does not accumulate during the time the cells are cultured. In various embodiments, the cells are cultured under limited glucose conditions for greater than or about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, or 70 hours. In various embodiments, the cells are cultured under limited glucose conditions for greater than or about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 95, or 100% of the total length of time the cells are cultured. While not intending to be bound by any particular theory, it is believed that limited glucose conditions may allow more favorable regulation of the cells.

In some embodiments, the cells are cultured in the presence of an excess of glucose. In particular embodiments, the amount of glucose that is added is greater than about 105% (such as about or greater than 110, 120, 150, 175, 200, 250, 300, 400, or 500%) or more of the amount of glucose that is consumed by the cells during a specific period of time. In some embodiments, glucose accumulates during the time the cells are cultured.

Exemplary lipids are any substance containing one or more fatty acids that are C4 and above fatty acids that are saturated, unsaturated, or branched.

Exemplary oils are lipids that are liquid at room temperature. In some embodiments, the lipid contains one or more C4 or above fatty acids (e.g., contains one or more saturated, unsaturated, or branched fatty acid with four or more carbons). In some embodiments, the oil is obtained from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, linseed, oleagineous microbial cells, Chinese tallow, or any combination of two or more of the foregoing.

Exemplary fatty acids include compounds of the formula RCOOH, where “R” is a hydrocarbon. Exemplary unsaturated fatty acids include compounds where “R” includes at least one carbon-carbon double bond. Exemplary unsaturated fatty acids include, but are not limited to, oleic acid, vaccenic acid, linoleic acid, palmitoleic acid, and arachidonic acid. Exemplary polyunsaturated fatty acids include compounds where “R” includes a plurality of carbon-carbon double bonds. Exemplary saturated fatty acids include compounds where “R” is a saturated aliphatic group. In some embodiments, the carbon source includes one or more C₁₂-C₂₂ fatty acids, such as a C₁₂ saturated fatty acid, a C₁₄ saturated fatty acid, a C₁₆ saturated fatty acid, a C₁₈ saturated fatty acid, a C₂₀ saturated fatty acid, or a C₂₂ saturated fatty acid. In an exemplary embodiment, the fatty acid is palmitic acid. In some embodiments, the carbon source is a salt of a fatty acid (e.g., an unsaturated fatty acid), a derivative of a fatty acid (e.g., an unsaturated fatty acid), or a salt of a derivative of fatty acid (e.g., an unsaturated fatty acid). Suitable salts include, but are not limited to, lithium salts, potassium salts, sodium salts, and the like. Di- and triglycerols are fatty acid esters of glycerol.

In some embodiments, the concentration of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride is at least or about 1 gram per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such as at least or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, 400, or more g/L. In some embodiments, the concentration of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride is between about 10 and about 400 g/L, such as between about 25 and about 300 g/L, between about 60 and about 180 g/L, or between about 75 and about 150 g/L. In some embodiments, the concentration includes the total amount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride that is added before and/or during the culturing of the host cells. In some embodiments, the carbon source includes both (i) a lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride and (ii) a carbohydrate, such as glucose. In some embodiments, the ratio of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride to the carbohydrate is about 1:1 on a carbon basis (i.e., one carbon in the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride per carbohydrate carbon). In particular embodiments, the amount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride is between about 60 and 180 g/L, and the amount of the carbohydrate is between about 120 and 360 g/L.

Exemplary microbial polypeptide carbon sources include one or more polypeptides from yeast or bacteria. Exemplary plant polypeptide carbon sources include one or more polypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.

Exemplary renewable carbon sources include cheese whey permeate, cornsteep liquor, sugar beet molasses, barley malt, and components from any of the foregoing. Exemplary renewable carbon sources also include glucose, hexose, pentose and xylose present in biomass, such as corn, switchgrass, sugar cane, cell waste of fermentation processes, and protein by-product from the milling of soy, corn, or wheat. In some embodiments, the biomass carbon source is a lignocellulosic, hemicellulosic, or cellulosic material such as, but are not limited to, a grass, wheat, wheat straw, bagasse, sugar cane bagasse, soft wood pulp, corn, corn cob or husk, corn kernel, fiber from corn kernels, corn stover, switch grass, rice hull product, or a by-product from wet or dry milling of grains (e.g., corn, sorghum, rye, triticate, barley, wheat, and/or distillers grains). Exemplary cellulosic materials include wood, paper and pulp waste, herbaceous plants, and fruit pulp. In some embodiments, the carbon source includes any plant part, such as stems, grains, roots, or tubers. In some embodiments, all or part of any of the following plants are used as a carbon source: corn, wheat, rye, sorghum, triticate, rice, millet, barley, cassava, legumes, such as beans and peas, potatoes, sweet potatoes, bananas, sugarcane, and/or tapioca. In some embodiments, the carbon source is a biomass hydrolysate, such as a biomass hydrolysate that includes both xylose and glucose or that includes both sucrose and glucose.

In some embodiments, the renewable carbon source (such as biomass) is pretreated before it is added to the cell culture medium. In some embodiments, the pretreatment includes enzymatic pretreatment, chemical pretreatment, or a combination of both enzymatic and chemical pretreatment (see, for example, Farzaneh et al., Bioresource Technology 96 (18): 2014-2018, 2005; U.S. Pat. No. 6,176,176; U.S. Pat. No. 6,106,888; which are each hereby incorporated by reference in their entireties, particularly with respect to the pretreatment of renewable carbon sources). In some embodiments, the renewable carbon source is partially or completely hydrolyzed before it is added to the cell culture medium.

In some embodiments, the renewable carbon source (such as corn stover) undergoes ammonia fiber expansion (AFEX) pretreatment before it is added to the cell culture medium (see, for example, Farzaneh et al., Bioresource Technology 96 (18): 2014-2018, 2005). During AFEX pretreatment, a renewable carbon source is treated with liquid anhydrous ammonia at moderate temperatures (such as about 60 to about 100° C.) and high pressure (such as about 250 to about 300 psi) for about 5 minutes. Then, the pressure is rapidly released. In this process, the combined chemical and physical effects of lignin solubilization, hemicellulose hydrolysis, cellulose decrystallization, and increased surface area enables near complete enzymatic conversion of cellulose and hemicellulose to fermentable sugars. AFEX pretreatment has the advantage that nearly all of the ammonia can be recovered and reused, while the remaining serves as nitrogen source for microbes in downstream processes. Also, a wash stream is not required for AFEX pretreatment. Thus, dry matter recovery following the AFEX treatment is essentially 100%. AFEX is basically a dry to dry process. The treated renewable carbon source is stable for long periods and can be fed at very high solid loadings in enzymatic hydrolysis or fermentation processes. Cellulose and hemicellulose are well preserved in the AFEX process, with little or no degradation. There is no need for neutralization prior to the enzymatic hydrolysis of a renewable carbon source that has undergone AFEX pretreatment. Enzymatic hydrolysis of AFEX-treated carbon sources produces clean sugar streams for subsequent fermentation use.

In some embodiments, the concentration of the carbon source (e.g., a renewable carbon source) is equivalent to at least or about 0.1, 0.5, 1, 1.5 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50% glucose (w/v). The equivalent amount of glucose can be determined by using standard HPLC methods with glucose as a reference to measure the amount of glucose generated from the carbon source. In some embodiments, the concentration of the carbon source (e.g., a renewable carbon source) is equivalent to between about 0.1 and about 20% glucose, such as between about 0.1 and about 10% glucose, between about 0.5 and about 10% glucose, between about 1 and about 10% glucose, between about 1 and about 5% glucose, or between about 1 and about 2% glucose.

In some embodiments, the carbon source includes yeast extract or one or more components of yeast extract. In some embodiments, the concentration of yeast extract is at least 1 gram of yeast extract per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such at least or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, or more g/L. In some embodiments, the concentration of yeast extract is between about 1 and about 300 g/L, such as between about 1 and about 200 g/L, between about 5 and about 200 g/L, between about 5 and about 100 g/L, or between about 5 and about 60 g/L. In some embodiments, the concentration includes the total amount of yeast extract that is added before and/or during the culturing of the host cells. In some embodiments, the carbon source includes both yeast extract (or one or more components thereof) and another carbon source, such as glucose. In some embodiments, the ratio of yeast extract to the other carbon source is about 1:5, about 1:10, or about 1:20 (w/w).

Additionally the carbon source may also be one-carbon substrates such as carbon dioxide, or methanol. Glycerol production from single carbon sources (e.g., methanol, formaldehyde, or formate) has been reported in methylotrophic yeasts (Yamada et al., Agric. Biol. Chem., 53(2) 541-543, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources) and in bacteria (Hunter et. al., Biochemistry, 24, 4148-4155, 1985, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). These organisms can assimilate single carbon compounds, ranging in oxidation state from methane to formate, and produce glycerol. The pathway of carbon assimilation can be through ribulose monophosphate, through serine, or through xylulose-momophosphate (Gottschalk, Bacterial Metabolism, Second Edition, Springer-Verlag: New York, 1986, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). The ribulose monophosphate pathway involves the condensation of formate with ribulose-5-phosphate to form a six carbon sugar that becomes fructose and eventually the three carbon product glyceraldehyde-3-phosphate. Likewise, the serine pathway assimilates the one-carbon compound into the glycolytic pathway via methylenetetrahydrofolate.

In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7^(th) ed., 415-32. Editors: Murrell et al., Publisher: Intercept, Andover, UK, 1993, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). Similarly, various species of Candida metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153(5), 485-9, 1990, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources).

In some embodiments, cells are cultured in a standard medium containing physiological salts and nutrients (see, e.g., Pourquie, J. et al., Biochemistry and Genetics of Cellulose Degradation, eds. Aubert et al., Academic Press, pp. 71-86, 1988 and Ilmen et al., Appl. Environ. Microbiol. 63:1298-1306, 1997, which are each hereby incorporated by reference in their entireties, particularly with respect to cell medias). Exemplary growth media are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of particular host cells are known by someone skilled in the art of microbiology or fermentation science.

In addition to an appropriate carbon source, the cell medium desirably contains suitable minerals, salts, cofactors, buffers, and other components known to those skilled in the art suitable for the growth of the cultures or the enhancement of isoprene production (see, for example, WO 2004/033646 and references cited therein and WO 96/35796 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect cell medias and cell culture conditions). In some embodiments where an isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acid is under the control of an inducible promoter, the inducing agent (e.g., a sugar, metal salt or antimicrobial), is desirably added to the medium at a concentration effective to induce expression of an isoprene synthase, DXS, IDI, and/or MVA pathway polypeptide. In some embodiments, cell medium has an antibiotic (such as kanamycin) that corresponds to the antibiotic resistance nucleic acid (such as a kanamycin resistance nucleic acid) on a vector that has one or more DXS, IDI, or MVA pathway nucleic acids.

Exemplary Cell Culture Conditions

Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Exemplary techniques may be found in Manual of Methods for General Bacteriology Gerhardt et al., eds), American Society for Microbiology, Washington, D.C. (1994) or Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., which are each hereby incorporated by reference in their entireties, particularly with respect to cell culture techniques. In some embodiments, the cells are cultured in a culture medium under conditions permitting the expression of one or more isoprene synthase, DXS, IDI, or MVA pathway polypeptides encoded by a nucleic acid inserted into the host cells.

Standard cell culture conditions can be used to culture the cells (see, for example, WO 2004/033646 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to cell culture and fermentation conditions). Cells are grown and maintained at an appropriate temperature, gas mixture, and pH (such as at about 20° C. to about 37° C., at about 6% to about 84% CO₂, and at a pH between about 5 to about 9). In some embodiments, cells are grown at 35° C. in an appropriate cell medium. In some embodiments, e.g., cultures are cultured at approximately 28° C. in appropriate medium in shake cultures or fermentors until the desired amount of isoprene and hydrogen co-production is achieved. In some embodiments, the pH ranges for fermentation are between about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH 8.0 or about 6.5 to about 7.0). Reactions may be performed under aerobic, anoxic, or anaerobic conditions based on the requirements of the host cells. In some embodiments, the cells are cultured under oxygen-limited conditions. In some embodiments, the cells are cultured in the presence of oxygen under conditions where 0.5 moles of oxygen are taken up per mole of isoprene produced. In some embodiments, the cells are cultured under anaerobic conditions. Exemplary culture conditions for a given filamentous fungus are known in the art and may be found in the scientific literature and/or from the source of the fungi such as the American Type Culture Collection and Fungal Genetics Stock Center.

In various embodiments, the cells are grown using any known mode of fermentation, such as batch, fed-batch, or continuous processes. In some embodiments, a batch method of fermentation is used. Classical batch fermentation is a closed system where the composition of the media is set at the beginning of the fermentation and is not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the cell medium is inoculated with the desired host cells and fermentation is permitted to occur adding nothing to the system. Typically, however, “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly until the time the fermentation is stopped. Within batch cultures, cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. In some embodiments, cells in log phase are responsible for the bulk of the isoprene production. In some embodiments, cells in stationary phase produce isoprene.

In some embodiments, a variation on the standard batch system is used, such as the Fed-Batch system. Fed-Batch fermentation processes comprise a typical batch system with the exception that the carbon source is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of carbon source in the cell medium. Fed-batch fermentations may be performed with the carbon source (e.g., glucose) in a limited or excess amount. Measurement of the actual carbon source concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen, and the partial pressure of waste gases such as CO₂. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., which is hereby incorporated by reference in its entirety, particularly with respect to cell culture and fermentation conditions.

In some embodiments, continuous fermentation methods are used. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or isoprene production. For example, one method maintains a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allows all other parameters to moderate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration (e.g., the concentration measured by media turbidity) is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, the cell loss due to media being drawn off is balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., which is hereby incorporated by reference in its entirety, particularly with respect to cell culture and fermentation conditions.

In some embodiments, cells are immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for isoprene production.

In some embodiments, bottles of liquid culture are placed in shakers in order to introduce oxygen to the liquid and maintain the uniformity of the culture. In some embodiments, an incubator is used to control the temperature, humidity, shake speed, and/or other conditions in which a culture is grown. The simplest incubators are insulated boxes with an adjustable heater, typically going up to ˜65° C. More elaborate incubators can also include the ability to lower the temperature (via refrigeration), or the ability to control humidity or CO₂ levels. Most incubators include a timer; some can also be programmed to cycle through different temperatures, humidity levels, etc. Incubators can vary in size from tabletop to units the size of small rooms.

If desired, a portion or all of the cell medium can be changed to replenish nutrients and/or avoid the build up of potentially harmful metabolic byproducts and dead cells. In the case of suspension cultures, cells can be separated from the media by centrifuging or filtering the suspension culture and then resuspending the cells in fresh media. In the case of adherent cultures, the media can be removed directly by aspiration and replaced. In some embodiments, the cell medium allows at least a portion of the cells to divide for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture (such as a continuous culture without dilution).

In some embodiments, a constitutive or leaky promoter (such as a Trc promoter) is used and a compound (such as IPTG) is not added to induce expression of the isoprene synthase, DXS, IDI, or MVA pathway nucleic acid(s) operably linked to the promoter. In some embodiments, a compound (such as IPTG) is added to induce expression of the isoprene synthase, DXS, IDI, or MVA pathway nucleic acid(s) operably linked to the promoter.

Exemplary Methods for Decoupling Isoprene Production from Cell Growth

Desirably, carbon from the feedstock is converted to isoprene rather than to the growth and maintenance of the cells. In some embodiments, the cells are grown to a low to medium OD₆₀₀, then production of isoprene is started or increased. This strategy permits a large portion of the carbon to be converted to isoprene.

In some embodiments, cells reach an optical density such that they no longer divide or divide extremely slowly, but continue to make isoprene for several hours (such as about 2, 4, 6, 8, 10, 15, 20, 25, 30, or more hours). For example, FIGS. 60A-67C illustrate that cells may continue to produce a substantial amount of mevalonic acid or isoprene after the cells reach an optical density such that they no longer divide or divide extremely slowly. In some cases, the optical density at 550 nm decreases over time (such as a decrease in the optical density after the cells are no longer in an exponential growth phase due to cell lysis), and the cells continue to produce a substantial amount of mevalonic acid or isoprene. In some embodiments, the optical density at 550 nm of the cells increases by less than or about 50% (such as by less than or about 40, 30, 20, 10, 5, or 0%) over a certain time period (such as greater than or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cells produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/g_(wcm)/hr) during this time period. In some embodiments, the amount of isoprene is between about 2 to about 5,000 nmole/g_(wcm)/hr, such as between about 2 to about 100 nmole/g_(wcm)/hr, about 100 to about 500 nmole/g_(wcm)/hr, about 150 to about 500 nmole/g_(wcm)/hr, about 500 to about 1,000 nmole/g_(wcm)/hr, about 1,000 to about 2,000 nmole/g_(wcm)/hr, or about 2,000 to about 5,000 nmole/g_(wcm)/hr. In some embodiments, the amount of isoprene is between about 20 to about 5,000 nmole/g_(wcm)/hr, about 100 to about 5,000 nmole/g_(wcm)/hr, about 200 to about 2,000 nmole/g_(wcm)/hr, about 200 to about 1,000 nmole/g_(wcm)/hr, about 300 to about 1,000 nmole/g_(wcm)/hr, or about 400 to about 1,000 nmole/g_(wcm)/hr.

In some embodiments, the optical density at 550 nm of the cells increases by less than or about 50% (such as by less than or about 40, 30, 20, 10, 5, or 0%) over a certain time period (such as greater than or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cells produce a cumulative titer (total amount) of isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/L of broth (mg/L_(broth), wherein the volume of broth includes the volume of the cells and the cell medium) during this time period. In some embodiments, the amount of isoprene is between about 2 to about 5,000 mg/L_(broth), such as between about 2 to about 100 mg/L_(broth), about 100 to about 500 mg/L_(broth), about 500 to about 1,000 mg/L_(broth), about 1,000 to about 2,000 mg/L_(broth), or about 2,000 to about 5,000 mg/L_(broth). In some embodiments, the amount of isoprene is between about 20 to about 5,000 mg/L_(broth), about 100 to about 5,000 mg/L_(broth), about 200 to about 2,000 mg/L_(broth), about 200 to about 1,000 mg/L_(broth), about 300 to about 1,000 mg/L_(broth), or about 400 to about 1,000 mg/L_(broth).

In some embodiments, the optical density at 550 nm of the cells increases by less than or about 50% (such as by less than or about 40, 30, 20, 10, 5, or 0%) over a certain time period (such as greater than or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cells convert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culture medium into isoprene during this time period. In some embodiments, the percent conversion of carbon into isoprene is between such as about 0.002 to about 4.0%, about 0.002 to about 3.0%, about 0.002 to about 2.0%, about 0.002 to about 1.6%, about 0.002 to about 0.005%, about 0.005 to about 0.01%, about 0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15 to about 0.2%, about 0.2 to about 0.3%, about 0.3 to about 0.5%, about 0.5 to about 0.8%, about 0.8 to about 1.0%, or about 1.0 to about 1.6%. In some embodiments, the percent conversion of carbon into isoprene is between about 0.002 to about 0.4%, 0.002 to about 0.16%, 0.04 to about 0.16%, about 0.005 to about 0.3%, about 0.01 to about 0.3%, or about 0.05 to about 0.3%.

In some embodiments, isoprene is only produced in stationary phase. In some embodiments, isoprene is produced in both the growth phase and stationary phase. In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD₆₀₀) during stationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amount of isoprene produced during the growth phase for the same length of time. In various embodiments, greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99% or more of the total amount of isoprene that is produced (such as the production of isoprene during a fermentation for a certain amount of time, such as 20 hours) is produced while the cells are in stationary phase. In various embodiments, greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99% or more of the total amount of isoprene that is produced (such as the production of isoprene during a fermentation for a certain amount of time, such as 20 hours) is produced while the cells divide slowly or not at all such that the optical density at 550 nm of the cells increases by less than or about 50% (such as by less than or about 40, 30, 20, 10, 5, or 0%). In some embodiments, isoprene is only produced in the growth phase.

In some embodiments, one or more MVA pathway, IDI, DXP, or isoprene synthase nucleic acids are placed under the control of a promoter or factor that is more active in stationary phase than in the growth phase. For example, one or more MVA pathway, IDI, DXP, or isoprene synthase nucleic acids may be placed under control of a stationary phase sigma factor, such as RpoS. In some embodiments, one or more MVA pathway, IDI, DXP, or isoprene synthase nucleic acids are placed under control of a promoter inducible in stationary phase, such as a promoter inducible by a response regulator active in stationary phase.

Production of Isoprene Within Safe Operating Ranges

The production of isoprene within safe operating levels according to its flammability characteristics simplifies the design and construction of commercial facilities, vastly improves the ability to operate safely, and limits the potential for fires to occur. In particular, the optimal ranges for the production of isoprene are within the safe zone, i.e., the nonflammable range of isoprene concentrations. In one such aspect, described herein is a method for the production of isoprene within the nonflammable range of isoprene concentrations (outside the flammability envelope of isoprene).

Thus, computer modeling and experimental testing were used to determine the flammability limits of isoprene (such as isoprene in the presence of O₂, N₂, CO₂, or any combination of two or more of the foregoing gases) in order to ensure process safety. The flammability envelope is characterized by the lower flammability limit (LFL), the upper flammability limit (UFL), the limiting oxygen concentration (LOC), and the limiting temperature. For a system to be flammable, a minimum amount of fuel (such as isoprene) must be in the presence of a minimum amount of oxidant, typically oxygen. The LFL is the minimum amount of isoprene that must be present to sustain burning, while the UFL is the maximum amount of isoprene that can be present. Above this limit, the mixture is fuel rich and the fraction of oxygen is too low to have a flammable mixture. The LOC indicates the minimum fraction of oxygen that must also be present to have a flammable mixture. The limiting temperature is based on the flash point of isoprene and is that lowest temperature at which combustion of isoprene can propagate. These limits are specific to the concentration of isoprene, type and concentration of oxidant, inerts present in the system, temperature, and pressure of the system. Compositions that fall within the limits of the flammability envelope propagate combustion and require additional safety precautions in both the design and operation of process equipment.

The following conditions were tested using computer simulation and mathematical analysis and experimental testing. If desired, other conditions (such as other temperature, pressure, and permanent gas compositions) may be tested using the methods described herein to determine the LFL, UFL, and LOC concentrations.

(1) Computer Simulation and Mathematical Analysis

Test Suite 1:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt %

N₂: 79 wt %-94 wt %

Test Suite 2:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt %

N₂: 79 wt %-94 wt %

Saturated with H₂O

Test Suite 3:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt %

N₂: 79 wt %-94 wt %

CO₂: 5 wt %-30 wt %

(2) Experimental Testing for Final Determination of Flammability Limits

Test Suite 1:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt %

N₂: 79 wt %-94 wt %

Test Suite 2:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt %

N₂: 79 wt %-94 wt %

Saturated with H₂O

Simulation software was used to give an estimate of the flammability characteristics of the system for several different testing conditions. CO₂ showed no significant affect on the system's flammability limits. Test suites 1 and 2 were confirmed by experimental testing. The modeling results were in-line with the experimental test results. Only slight variations were found with the addition of water.

The LOC was determined to be 9.5 vol % for an isoprene, O₂, N₂, and CO₂ mixture at 40° C. and 1 atmosphere. The addition of up to 30% CO₂ did not significantly affect the flammability characteristics of an isoprene, O₂, and N₂ mixture. Only slight variations in flammability characteristics were shown between a dry and water saturated isoprene, O₂, and N₂ system. The limiting temperature is about −54° C. Temperatures below about −54° C. are too low to propagate combustion of isoprene.

In some embodiments, the LFL of isoprene ranges from about 1.5 vol. % to about 2.0 vol %, and the UFL of isoprene ranges from about 2.0 vol. % to about 12.0 vol. %, depending on the amount of oxygen in the system. In some embodiments, the LOC is about 9.5 vol % oxygen. In some embodiments, the LFL of isoprene is between about 1.5 vol. % to about 2.0 vol %, the UFL of isoprene is between about 2.0 vol. % to about 12.0 vol. %, and the LOC is about 9.5 vol % oxygen when the temperature is between about 25° C. to about 55° C. (such as about 40° C.) and the pressure is between about 1 atmosphere and 3 atmospheres.

In some embodiments, isoprene is produced in the presence of less than about 9.5 vol % oxygen (that is, below the LOC required to have a flammable mixture of isoprene). In some embodiments in which isoprene is produced in the presence of greater than or about 9.5 vol % oxygen, the isoprene concentration is below the LFL (such as below about 1.5 vol. %). For example, the amount of isoprene can be kept below the LFL by diluting the isoprene composition with an inert gas (e.g., by continuously or periodically adding an inert gas such as nitrogen to keep the isoprene composition below the LFL). In some embodiments in which isoprene is produced in the presence of greater than or about 9.5 vol % oxygen, the isoprene concentration is above the UFL (such as above about 12 vol. %). For example, the amount of isoprene can be kept above the UFL by using a system (such as any of the cell culture systems described herein) that produces isoprene at a concentration above the UFL. If desired, a relatively low level of oxygen can be used so that the UFL is also relatively low. In this case, a lower isoprene concentration is needed to remain above the UFL.

In some embodiments in which isoprene is produced in the presence of greater than or about 9.5 vol % oxygen, the isoprene concentration is within the flammability envelope (such as between the LFL and the UFL). In some embodiments when the isoprene concentration may fall within the flammability envelope, one or more steps are performed to reduce the probability of a fire or explosion. For example, one or more sources of ignition (such as any materials that may generate a spark) can be avoided. In some embodiments, one or more steps are performed to reduce the amount of time that the concentration of isoprene remains within the flammability envelope. In some embodiments, a sensor is used to detect when the concentration of isoprene is close to or within the flammability envelope. If desired, the concentration of isoprene can be measured at one or more time points during the culturing of cells, and the cell culture conditions and/or the amount of inert gas can be adjusted using standard methods if the concentration of isoprene is close to or within the flammability envelope. In particular embodiments, the cell culture conditions (such as fermentation conditions) are adjusted to either decrease the concentration of isoprene below the LFL or increase the concentration of isoprene above the UFL. In some embodiments, the amount of isoprene is kept below the LFL by diluting the isoprene composition with an inert gas (such as by continuously or periodically adding an inert gas to keep the isoprene composition below the LFL).

In some embodiments, the amount of flammable volatiles other than isoprene (such as one or more sugars) is at least about 2, 5, 10, 50, 75, or 100-fold less than the amount of isoprene produced. In some embodiments, the portion of the gas phase other than isoprene gas comprises between about 0% to about 100% (volume) oxygen, such as between about 0% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 90% to about 90%, or about 90% to about 100% (volume) oxygen. In some embodiments, the portion of the gas phase other than isoprene gas comprises between about 0% to about 99% (volume) nitrogen, such as between about 0% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 90% to about 90%, or about 90% to about 99% (volume) nitrogen.

In some embodiments, the portion of the gas phase other than isoprene gas comprises between about 1% to about 50% (volume) CO₂, such as between about 1% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, or about 40% to about 50% (volume) CO₂.

In some embodiments, an isoprene composition also contains ethanol. For example, ethanol may be used for extractive distillation of isoprene, resulting in compositions (such as intermediate product streams) that include both ethanol and isoprene. Desirably, the amount of ethanol is outside the flammability envelope for ethanol. The LOC of ethanol is about 8.7 vol %, and the LFL for ethanol is about 3.3 vol % at standard conditions, such as about 1 atmosphere and about 6° F. (NFPA 69 Standard on Explosion Prevention Systems, 2008 edition, which is hereby incorporated by reference in its entirety, particularly with respect to LOC, LFL, and UFL values). In some embodiments, compositions that include isoprene and ethanol are produced in the presence of less than the LOC required to have a flammable mixture of ethanol (such as less than about 8.7% vol %). In some embodiments in which compositions that include isoprene and ethanol are produced in the presence of greater than or about the LOC required to have a flammable mixture of ethanol, the ethanol concentration is below the LFL (such as less than about 3.3 vol. %).

In various embodiments, the amount of oxidant (such as oxygen) is below the LOC of any fuel in the system (such as isoprene or ethanol). In various embodiments, the amount of oxidant (such as oxygen) is less than about 60, 40, 30, 20, 10, or 5% of the LOC of isoprene or ethanol. In various embodiments, the amount of oxidant (such as oxygen) is less than the LOC of isoprene or ethanol by at least 2, 4, 5, or more absolute percentage points (vol %). In particular embodiments, the amount of oxygen is at least 2 absolute percentage points (vol %) less than the LOC of isoprene or ethanol (such as an oxygen concentration of less than 7.5 vol % when the LOC of isoprene is 9.5 vol %). In various embodiments, the amount of fuel (such as isoprene or ethanol) is less than or about 25, 20, 15, 10, or 5% of the LFL for that fuel.

High Efficiency Production and Recovery of Isoprene, a Volatile Hydrocarbon, by Fermentation

Methods are provided herein of producing isoprene comprising a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the liquid phase concentration of isoprene is less than about 200 mg/L. In some embodiments, the liquid phase concentration of isoprene in the culture is less than about any of 175 mg/L, 150 mg/L, 125 mg/L, 100 mg/L, 75 mg/L, 50 mg/L, 25 mg/L, 20 mg/L, 15 mg/L, 10 mg/L, 5 mg/L, or 2.5 mg/L. In some embodiments, the liquid phase concentration of isoprene in culture is between about any of 0.1 mg/L to 200 mg/L, 1 mg/L to 200 mg/L, 1 mg/L to 150 mg/L, 1 mg/L to 100 mg/L, 1 mg/L to 50 mg/L, 1 mg/L to 25 mg/L, 1 mg/L to 20 mg/L, or 10 mg/L to 20 mg/L. In some embodiments, the isoprene produced is any concentration or amount disclosed in the section entitled “Exemplary Production of Isoprene.” In some embodiments, the liquid phase concentration is below the solubility limit of isoprene.

In some embodiments of the methods, the cells produce greater than about 400 nmole/g_(wcm)/hour of isoprene. In some embodiments, the amount of isoprene is between about any of 400 nmole/g_(wcm)/hour to 1 mole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 1 mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 40 mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 4 mmole/g_(wcm)/hour, 1 mmole/g_(wcm)/hour to 1.5 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour to 3 mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour to 5 mmole/g_(wcm)/hour, 5 mmole/g_(wcm)/hour to 25 mmole/g_(wcm)/hour, 25 mmole/g_(wcm)/hour to 100 mmole/g_(wcm)/hour, 100 mmole/g_(wcm)/hour to 500 mmole/g_(wcm)/hour, or 500 mmole/g_(wcm)/hour to 1000 mmole/g_(wcm)/hour. In some embodiments, the amount of isoprene is about any of 1 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour, 2 mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour, 4 mmole/g_(wcm)/hour, or 5 mmole/g_(wcm)/hour.

The low value for Henry's coefficient (in M atm⁻¹ units) means that isoprene can be recovered from fermentation broth by gas stripping at low sparging rates, for example 0.01 vvm to 2 vvm. In some embodiments, the gas sparging rate is between about any of 0.1 vvm to 1 vvm, 0.01 vvm to 0.5 vvm, 0.2 vvm to 1 vvm, or 0.5 vvm to 1 vvm. In some embodiments, the gas sparging rate is about any of 0.1 vvm, 0.25 vvm, 0.5 vvm, 0.75 vvm, 1 vvm, 1.25 vvm, 1.5 vvm, 1.75 vvm, or 2 vvm. In some embodiments, the low sparging rates are maintained for the entire course of the fermentation run, during growth phase, or during stationary phase. In some embodiments, the low sparging rates are maintained for between about any of 1 hour to 5 hours, 5 hours to 10 hours, 10 hours to 20 hours, 20 hours to 30 hours, 30 hours to 40 hours, 40 hours to 50 hours, or 50 hours to 60 hours. The lower desirable gas sparge limit is defined by the point at which the aqueous phase becomes saturated with isoprene and a liquid organic phase forms. This can only occur below the boiling point of isoprene (34.1° C. at 1 atm), above which a liquid isoprene phase will never form. At temperatures below the boiling point of isoprene, the formation of a liquid phase is determined by the aqueous solubility of isoprene, which is approximately 650 mg/L at 25° C. While it is highly desirable to avoid the formation of a liquid isoprene phase, it is not absolutely required provided that the cells can tolerate the presence of liquid isoprene without toxic effects.

In some embodiments, the oxygen, CO₂, and isoprene are any of the amounts or concentrations discussed in the section entitled “Production of Isoprene with Safe Operating Ranges.” In some embodiments, all the oxygen is consumed by the cells while maintaining fully aerobic metabolism. In some embodiments, an excess of oxygen is used in order to satisfy the oxygen demands of the cells. Desirable ranges of oxygen in the off-gas are less than 20%, or less than 15% or less than 10% (v/v). Levels of oxygen below the limiting oxygen concentration required for combustion of isoprene (9.5% v/v at 1 atm) are particularly desirable. In some embodiments, oxygen-enriched air is utilized with the purpose of allowing minimal gas sweep rates while satisfying the cellular oxygen demand. In some embodiments, the portion of the gas phase of the gas sweep comprises between about 0.1% to about 10%, about 10% to about 20%, or about 20% to about 30% (volume) oxygen. In some embodiments, isoprene fermentations are performed under high pressure in order minimize the amount of excess oxygen required to maintain the required dissolved oxygen levels in the liquid phase.

In some embodiments, the reduction of the gas sweep rate through the fermentor is advantageous for an integrated isoprene production process in that such conditions enrich the off-gas isoprene levels up to about 30,000 μg/L (about 1% v/v) without adversely affecting the physiology of the cells.

In some embodiments, reduced gas-sparge rates do not significantly adversely affect the physiology of the cells. In some embodiments, the carbon dioxide evolution rate of cells in culture with reduced gas-sparge rates is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour. In some embodiments, cell viability with reduced gas-sparge rates is reduced by less than about any of 1.75-fold, 1.5-fold, 1.25-fold, 1-fold, 0.75-fold, 0.5-fold, or 0.25-fold. In some embodiments, cell viability with reduced gas-sparge rates is reduced by about 2-fold. In some embodiments, cell viability with reduced gas-sparge rates of a cell expressing a MVA pathway and/or DXP pathway RNA and/or protein from one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid is compared to a control cell lacking one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid with reduced gas-sparge rates. In some embodiments, cell viability with reduced gas-sparge rates of a cell expressing a MVA pathway and/or DXP pathway RNA and/or protein from one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid under the control of an inducible promoter, wherein the promotor is induced, is compared to a control cell containing one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid under the control of an inducible promoter, wherein the promotor is not induced (uninduced) with reduced gas-sparge rates. In some embodiments, the inducible promoter is a beta-galactosidase promotor.

In some embodiments, the fermentation of a genetically modified host organism that converts at least 5% of the total carbon consumed by the organism into a volatile, unsaturated hydrocarbon. In some embodiments, the production of an unsaturated hydrocarbon at such a rate as to be present in the fermentation off-gas at a level of at least about any of 100 μg/L, 500 μg/L, 1000 μg/L, 2, 500 μg/L, 5,000 μg/L, 7,500 μg/L, or 10,000 μg/L.

In some embodiments, the unsaturated hydrocarbon is recovered from the off-gas stream in a manner that is suited to high-rates of production, which correspond to concentrations in the off-gas of at least about any of 100 μg/L, 500 μg/L, 1000 μg/L, 2,500 μg/L, 5,000 μg/L, 7,500 μg/L, or 10,000 μg/L. In some embodiments, the continuous extraction and recovery of an unsaturated hydrocarbon from the fermentation off-gas in particular at low gas sweep rates such that the resulting off-gas is enriched in the volatile component of interest. In some embodiments, recovery of the volatile hydrocarbon by methods that depend on elevated concentrations of the volatile. For example, efficient capture of isoprene in fermentation off-gas through the use of compression/condensation or extractive distillation technologies. Also contemplated is the use of activated carbon cartridges in addition to silica gel adsorbants, desorption and concentration of isoprene from carbon cartridges, and/or construction and fermentation of host organisms such as E. coli strains that can convert about 5% or more of the glucose substrate to isoprene and result in off-gas concentrations of greater than about 15,000 μg/L isoprene. Recovery methods include any of the methods described herein.

Also provided herein are methods of producing a compound, wherein the compound has one or more characteristics selected from the group consisting of (a) a Henry's law coefficient of less than about 250 M/atm and (b) a solubility in water of less than about 100 g/L. In some embodiments, the method comprises: a) culturing cells under suitable conditions for production of the compound, wherein gas is added (such as the addition of gas to a system such as a fermentation system) at a gas sparging rate between about 0.01 vvm to about 2 vvm; and b) producing the compound.

In some embodiments, the amount of the compound that partitions into the cell mass is not included in the liquid phase solubility values. In some embodiments, the liquid phase concentration is below the solubility limit of compound.

In some embodiments, the compounds can be continuously recovered from fermentation broth by gas stripping at moderate to low gas sparging rates, in particular those compounds with Henry's law coefficients of about any of less than 250 M/atm, 200 M/atm, 150 M/atm, 100 M/atm, 75 M/atm, 50 M/atm, 25 M/atm, 10 M/atm, 5 M/atm, or 1 M/atm. Examples include aldehydes such as acetaldehyde (15 M/atm), ketones such as acetone (30 M/atm) or 2-butanone (20 M/atm), or alcohols including methanol (220 M/atm), ethanol (200 M/atm), 1-butanol (120 M/atm) or C5 alcohols including 3-methyl-3-buten-1-ol, and 3-methyl-2-buten-1-ol (50-100 M/atm). Esters of alcohols generally have lower Henry's constants than the respective alcohols, for example ethyl acetate (6-9 M/atm) or the acetyl esters of C5 alcohols (<5 M/atm). Compounds with Henry's law coefficients of less than 1 M/atm are particularly desirable. Examples include hemiterpenes, monoterpenes, or sesquiterpenes, in addition to other hydrocarbons such as C1 to C5 hydrocarbons (e.g., methane, ethane, ethylene, or propylene). In some embodiments, the hydrocarbons such as C1 to C5 hydrocarbons are saturated, unsaturated, or branched.

In general, there is a correlation between Henry's law coefficient and water solubility in that compounds with very low coefficients are sparingly soluble in water (substantially water insoluble). Although volatiles with infinite solubilities in water (e.g. acetone or ethanol) can be removed by gas stripping, desirable solubility limits are less than about any of 100 g/L, 75 g/L, 50 g/L, 25 g/L, 10 g/L, 5 g/L, or 1 g/L.

In some embodiments of any of the methods of producing any of the compounds described above, the gas sparging rate is between about any of 0.1 vvm to 1 vvm, 0.2 vvm to 1 vvm, or 0.5 vvm to 1 vvm. In some embodiments, the gas sparging rate is about any of 0.1 vvm, 0.25 vvm, 0.5 vvm, 0.75 vvm, 1 vvm, 1.25 vvm, 1.5 vvm, 1.75 vvm, or 2 vvm. In some embodiments, the low sparging rates are maintained for the entire course of the fermentation run, during growth phase, or during stationary phase. In some embodiments, the low sparging rates are maintained for between about any of 1 hour to 5 hours, 5 hours to 10 hours, 10 hours to 20 hours, 20 hours to 30 hours, 30 hours to 40 hours, 40 hours to 50 hours, or 50 hours to 60 hours.

Any of the systems described herein can be used in the methods of producing a compound described above. Standard methods would be used to purify such as those described in the section entitled “Exemplary Purification Methods.” Separation can be performed post-recovery for example, by distillation or selective adsorption techniques.

Exemplary Production of BioIsoprene

In some embodiments, the cells are cultured in a culture medium under conditions permitting the production of isoprene by the cells.

By “peak absolute productivity” is meant the maximum absolute amount of isoprene in the off-gas during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By “peak absolute productivity time point” is meant the time point during a fermentation run when the absolute amount of isoprene in the off-gas is at a maximum during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). In some embodiments, the isoprene amount is measured at the peak absolute productivity time point. In some embodiments, the peak absolute productivity for the cells is about any of the isoprene amounts disclosed herein.

By “peak specific productivity” is meant the maximum amount of isoprene produced per cell during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By “peak specific productivity time point” is meant the time point during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run) when the amount of isoprene produced per cell is at a maximum. The peak specific productivity is determined by dividing the total productivity by the amount of cells, as determined by optical density at 600 nm (OD₆₀₀). In some embodiments, the isoprene amount is measured at the peak specific productivity time point. In some embodiments, the peak specific productivity for the cells is about any of the isoprene amounts per cell disclosed herein.

By “peak volumetric productivity” is meant the maximum amount of isoprene produced per volume of broth (including the volume of the cells and the cell medium) during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By “peak specific volumetric productivity time point” is meant the time point during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run) when the amount of isoprene produced per volume of broth is at a maximum. The peak specific volumetric productivity is determined by dividing the total productivity by the volume of broth and amount of time. In some embodiments, the isoprene amount is measured at the peak specific volumetric productivity time point. In some embodiments, the peak specific volumetric productivity for the cells is about any of the isoprene amounts per volume per time disclosed herein.

By “peak concentration” is meant the maximum amount of isoprene produced during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By “peak concentration time point” is meant the time point during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run) when the amount of isoprene produced per cell is at a maximum. In some embodiments, the isoprene amount is measured at the peak concentration time point. In some embodiments, the peak concentration for the cells is about any of the isoprene amounts disclosed herein.

By “average volumetric productivity” is meant the average amount of isoprene produced per volume of broth (including the volume of the cells and the cell medium) during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). The average volumetric productivity is determined by dividing the total productivity by the volume of broth and amount of time. In some embodiments, the average specific volumetric productivity for the cells is about any of the isoprene amounts per volume per time disclosed herein.

By “cumulative total productivity” is meant the cumulative, total amount of isoprene produced during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). In some embodiments, the cumulative, total amount of isoprene is measured. In some embodiments, the cumulative total productivity for the cells is about any of the isoprene amounts disclosed herein.

As used herein, “relative detector response” refers to the ratio between the detector response (such as the GC/MS area) for one compound (such as isoprene) to the detector response (such as the GC/MS area) of one or more compounds (such as all C5 hydrocarbons). The detector response may be measured as described herein, such as the GC/MS analysis performed with an Agilent 6890 GC/MS system fitted with an Agilent HP-5MS GC/MS column (30 m×250 μm; 0.25 μm film thickness). If desired, the relative detector response can be converted to a weight percentage using the response factors for each of the compounds. This response factor is a measure of how much signal is generated for a given amount of a particular compound (that is, how sensitive the detector is to a particular compound). This response factor can be used as a correction factor to convert the relative detector response to a weight percentage when the detector has different sensitivities to the compounds being compared. Alternatively, the weight percentage can be approximated by assuming that the response factors are the same for the compounds being compared. Thus, the weight percentage can be assumed to be approximately the same as the relative detector response.

In some embodiments, the cells in culture produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 12,500, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000, 125,000, 150,000, 188,000, or more nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/g_(wcm)/hr). In some embodiments, the amount of isoprene is between about 2 to about 200,000 nmole/g_(wcm)/hr, such as between about 2 to about 100 nmole/g_(wcm)/hr, about 100 to about 500 nmole/g_(wcm)/hr, about 150 to about 500 nmole/g_(wcm)/hr, about 500 to about 1,000 nmole/g_(wcm)/hr, about 1,000 to about 2,000 nmole/g_(wcm)/hr, or about 2,000 to about 5,000 nmole/g_(wcm)/hr, about 5,000 to about 10,000 nmole/g_(wcm)/hr, about 10,000 to about 50,000 nmole/g_(wcm)/hr, about 50,000 to about 100,000 nmole/g_(wcm)/hr, about 100,000 to about 150,000 nmole/g_(wcm)/hr, or about 150,000 to about 200,000 nmole/g_(wcm)/hr. In some embodiments, the amount of isoprene is between about 20 to about 5,000 nmole/g_(wcm)/hr, about 100 to about 5,000 nmole/g_(wcm)/hr, about 200 to about 2,000 nmole/g_(wcm)/hr, about 200 to about 1,000 nmole/g_(wcm)/hr, about 300 to about 1,000 nmole/g_(wcm)/hr, or about 400 to about 1,000 nmole/g_(wcm)/hr, about 1,000 to about 5,000 nmole/g_(wcm)/hr, about 2,000 to about 20,000 nmole/g_(wcm)/hr, about 5,000 to about 50,000 nmole/g_(wcm)/hr, about 10,000 to about 100,000 nmole/g_(wcm)/hr, about 20,000 to about 150,000 nmole/g_(wcm)/hr, or about 20,000 to about 200,000 nmole/g_(wcm)/hr.

The amount of isoprene in units of nmole/g_(wcm)/hr can be measured as disclosed in U.S. Pat. No. 5,849,970, which is hereby incorporated by reference in its entirety, particularly with respect to the measurement of isoprene production. For example, two mL of headspace (e.g., headspace from a culture such as 2 mL of culture cultured in sealed vials at 32° C. with shaking at 200 rpm for approximately 3 hours) are analyzed for isoprene using a standard gas chromatography system, such as a system operated isothermally (85° C.) with an n-octane/porasil C column (Alltech Associates, Inc., Deerfield, Ill.) and coupled to a RGD2 mercuric oxide reduction gas detector (Trace Analytical, Menlo Park, Calif.) (see, for example, Greenberg et al, Atmos. Environ. 27A: 2689-2692, 1993; Silver et al., Plant Physiol. 97:1588-1591, 1991, which are each hereby incorporated by reference in their entireties, particularly with respect to the measurement of isoprene production). The gas chromatography area units are converted to nmol isoprene via a standard isoprene concentration calibration curve. In some embodiments, the value for the grams of cells for the wet weight of the cells is calculated by obtaining the A₆₀₀ value for a sample of the cell culture, and then converting the A₆₀₀ value to grams of cells based on a calibration curve of wet weights for cell cultures with a known A₆₀₀ value. In some embodiments, the grams of the cells is estimated by assuming that one liter of broth (including cell medium and cells) with an A₆₀₀ value of 1 has a wet cell weight of 1 gram. The value is also divided by the number of hours the culture has been incubating for, such as three hours.

In some embodiments, the cells in culture produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wet weight of the cells/hr (ng/g_(wcm)/h). In some embodiments, the amount of isoprene is between about 2 to about 5,000 ng/g_(wcm)/h, such as between about 2 to about 100 ng/g_(wcm)/h, about 100 to about 500 ng/g_(wcm)/h, about 500 to about 1,000 ng/g_(wcm)/h, about 1,000 to about 2,000 ng/g_(wcm)/h, or about 2,000 to about 5,000 ng/g_(wcm)/h. In some embodiments, the amount of isoprene is between about 20 to about 5,000 ng/g_(wcm)/h, about 100 to about 5,000 ng/g_(wcm)/h, about 200 to about 2,000 ng/g_(wcm)/h, about 200 to about 1,000 ng/g_(wcm)/h, about 300 to about 1,000 ng/g_(wcm)/h, or about 400 to about 1,000 ng/g_(wcm)/h. The amount of isoprene in ng/g_(wcm)/h can be calculated by multiplying the value for isoprene production in the units of nmole/g_(wcm)/hr discussed above by 68.1 (as described in Equation 5 below).

In some embodiments, the cells in culture produce a cumulative titer (total amount) of isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/L of broth (mg/L_(broth), wherein the volume of broth includes the volume of the cells and the cell medium). In some embodiments, the amount of isoprene is between about 2 to about 5,000 mg/L_(broth), such as between about 2 to about 100 mg/L_(broth), about 100 to about 500 mg/L_(broth), about 500 to about 1,000 mg/L_(broth), about 1,000 to about 2,000 mg/L_(broth), or about 2,000 to about 5,000 mg/L_(broth). In some embodiments, the amount of isoprene is between about 20 to about 5,000 mg/L_(broth), about 100 to about 5,000 mg/L_(broth), about 200 to about 2,000 mg/L_(broth), about 200 to about 1,000 mg/L_(broth), about 300 to about 1,000 mg/L_(broth), or about 400 to about 1,000 mg/L_(broth).

The specific productivity of isoprene in mg of isoprene/L of headspace from shake flask or similar cultures can be measured by taking a 1 ml sample from the cell culture at an OD₆₀₀ value of approximately 1.0, putting it in a 20 mL vial, incubating for 30 minutes, and then measuring the amount of isoprene in the headspace (as described, for example, in Example I, part II). If the OD₆₀₀ value is not 1.0, then the measurement can be normalized to an OD₆₀₀ value of 1.0 by dividing by the OD₆₀₀ value. The value of mg isoprene/L headspace can be converted to mg/L_(broth)/hr/OD₆₀₀ of culture broth by multiplying by a factor of 38. The value in units of mg/L_(broth)/hr/OD₆₀₀ can be multiplied by the number of hours and the OD₆₀₀ value to obtain the cumulative titer in units of mg of isoprene/L of broth.

In some embodiments, the cells in culture have an average volumetric productivity of isoprene at greater than or about 0.1, 1.0, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100, 1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, or more mg of isoprene/L of broth/hr (mg/L_(broth)/hr, wherein the volume of broth includes the volume of the cells and the cell medium). In some embodiments, the average volumetric productivity of isoprene is between about 0.1 to about 3,500 mg/L_(broth)/hr, such as between about 0.1 to about 100 mg/L_(broth)/hr, about 100 to about 500 mg/L_(broth)/hr, about 500 to about 1,000 mg/L_(broth)/hr, about 1,000 to about 1,500 mg/L_(broth)/hr, about 1,500 to about 2,000 mg/L_(broth)/hr, about 2,000 to about 2,500 mg/L_(broth)/hr, about 2,500 to about 3,000 mg/L_(broth)/hr, or about 3,000 to about 3,500 mg/L_(broth)/hr. In some embodiments, the average volumetric productivity of isoprene is between about 10 to about 3,500 mg/L_(broth)/hr, about 100 to about 3,500 mg/L_(broth)/hr, about 200 to about 1,000 mg/L_(broth)/hr, about 200 to about 1,500 mg/L_(broth)/hr, about 1,000 to about 3,000 mg/L_(broth)/hr, or about 1,500 to about 3,000 mg/L_(broth)/hr.

In some embodiments, the cells in culture have a peak volumetric productivity of isoprene at greater than or about 0.5, 1.0, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100, 1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,750, 4,000, 4,250, 4,500, 4,750, 5,000, 5,250, 5,500, 5,750, 6,000, 6,250, 6,500, 6,750, 7,000, 7,250, 7,500, 7,750, 8,000, 8,250, 8,500, 8,750, 9,000, 9,250, 9,500, 9,750, 10,000, 12,500, 15,000, or more mg of isoprene/L of broth/hr (mg/L_(broth)/hr, wherein the volume of broth includes the volume of the cells and the cell medium). In some embodiments, the peak volumetric productivity of isoprene is between about 0.5 to about 15,000 mg/L_(broth)/hr, such as between about 0.5 to about 10 mg/L_(broth)/hr, about 1.0 to about 100 mg/L_(broth)/hr, about 100 to about 500 mg/L_(broth)/hr, about 500 to about 1,000 mg/L_(broth)/hr, about 1,000 to about 1,500 mg/L_(broth)/hr, about 1,500 to about 2,000 mg/L_(broth)/hr, about 2,000 to about 2,500 mg/L_(broth)/hr, about 2,500 to about 3,000 mg/L_(broth)/hr, about 3,000 to about 3,500 mg/L_(broth)/hr, about 3,500 to about 5,000 mg/L_(broth)/hr, about 5,000 to about 7,500 mg/L_(broth)/hr, about 7,500 to about 10,000 mg/L_(broth)/hr, about 10,000 to about 12,500 mg/L_(broth)/h, or about 12,500 to about 15,000 mg/L_(broth)/hr. In some embodiments, the peak volumetric productivity of isoprene is between about 10 to about 15,000 mg/L_(broth)/hr, about 100 to about 2,500 mg/L_(broth)/hr, about 1,000 to about 5,000 mg/L_(broth)/hr, about 2,500 to about 7,500 mg/L_(broth)/hr, about 5,000 to about 10,000 mg/L_(broth)/hr, about 7,500 to about 12,500 mg/L_(broth)/hr, or about 10,000 to about 15,000 mg/L_(broth)/hr.

The instantaneous isoprene production rate in mg/L_(broth)/hr in a fermentor can be measured by taking a sample of the fermentor off-gas, analyzing it for the amount of isoprene (in units such as mg of isoprene per L_(gas)) as described, for example, in Example I, part II and multiplying this value by the rate at which off-gas is passed though each liter of broth (e.g., at 1 vvm (volume of air/volume of broth/minute) this is 60 L_(gas) per hour). Thus, an off-gas level of 1 mg/L_(gas) corresponds to an instantaneous production rate of 60 mg/L_(broth)/hr at air flow of 1 vvm. If desired, the value in the units mg/L_(broth)/hr can be divided by the OD₆₀₀ value to obtain the specific rate in units of mg/L_(broth)/hr/OD. The average value of mg isoprene/L_(gas) can be converted to the total product productivity (grams of isoprene per liter of fermentation broth, mg/L_(broth)) by multiplying this average off-gas isoprene concentration by the total amount of off-gas sparged per liter of fermentation broth during the fermentation. Thus, an average off-gas isoprene concentration of 0.5 mg/L_(broth)/hr over 10 hours at 1 vvm corresponds to a total product concentration of 300 mg isoprene/L_(broth).

In some embodiments, the cells in culture convert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 23.2, 23.4, 23.6, 23.8, 24.0, 25.0, 30.0, 31.0, 32.0, 33.0, 35.0, 37.5, 40.0, 45.0, 47.5, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, or 90.0 molar % of the carbon in the cell culture medium into isoprene. In some embodiments, the percent conversion of carbon into isoprene is between about 0.002 to about 90.0 molar %, such as about 0.002 to about 0.005%, about 0.005 to about 0.01%, about 0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15 to about 0.2%, about 0.2 to about 0.3%, about 0.3 to about 0.5%, about 0.5 to about 0.8%, about 0.8 to about 1.0%, about 1.0 to about 1.6%, about 1.6 to about 3.0%, about 3.0 to about 5.0%, about 5.0 to about 8.0%, about 8.0 to about 10.0%, about 10.0 to about 15.0%, about 15.0 to about 20.0%, about 20.0 to about 25.0%, about 25.0% to 30.0%, about 30.0% to 35.0%, about 35.0% to 40.0%, about 45.0% to 50.0%, about 50.0% to 55.0%, about 55.0% to 60.0%, about 60.0% to 65.0%, about 65.0% to 70.0%, about 75.0% to 80.0%, about 80.0% to 85.0%, or about 85.0% to 90.0%. In some embodiments, the percent conversion of carbon into isoprene is between about 0.002 to about 0.4 molar %, 0.002 to about 0.16 molar %, 0.04 to about 0.16 molar %, about 0.005 to about 0.3 molar %, about 0.01 to about 0.3 molar %, about 0.05 to about 0.3 molar %, about 0.1 to 0.3 molar %, about 0.3 to about 1.0 molar %, about 1.0 to about 5.0 molar %, about 2 to about 5.0 molar %, about 5.0 to about 10.0 molar %, about 7 to about 10.0 molar %, about 10.0 to about 20.0 molar %, about 12 to about 20.0 molar %, about 16 to about 20.0 molar %, about 18 to about 20.0 molar %, about 18 to 23.2 molar %, about 18 to 23.6 molar %, about 18 to about 23.8 molar %, about 18 to about 24.0 molar %, about 18 to about 25.0 molar %, about 20 to about 30.0 molar %, about 30 to about 40.0 molar %, about 30 to about 50.0 molar %, about 30 to about 60.0 molar %, about 30 to about 70.0 molar %, about 30 to about 80.0 molar %, or about 30 to about 90.0 molar %.

The percent conversion of carbon into isoprene (also referred to as “% carbon yield”) can be measured by dividing the moles carbon in the isoprene produced by the moles carbon in the carbon source (such as the moles of carbon in batched and fed glucose and yeast extract). This number is multiplied by 100% to give a percentage value (as indicated in Equation 1). % Carbon Yield=(moles carbon in isoprene produced)/(moles carbon in carbon source)*100  Equation 1

For this calculation, yeast extract can be assumed to contain 50% w/w carbon. As an example, for the 500 liter described in Example 7, part VIII, the percent conversion of carbon into isoprene can be calculated as shown in Equation 2. % Carbon Yield=(39.1 g isoprene*1/68.1 mol/g*5 C/mol)/[(181221 g glucose*1/180 mol/g*6 C/mol)+(17780 g yeast extract*0.5*1/12 mol/g)]*100=0.042%  Equation 2

For the two 500 liter fermentations described herein (Example 7, parts VII and VIII), the percent conversion of carbon into isoprene was between 0.04-0.06%. A 0.11-0.16% carbon yield has been achieved using 14 liter systems as described herein. Example 11, part V describes the 1.53% conversion of carbon to isoprene using the methods described herein.

One skilled in the art can readily convert the rates of isoprene production or amount of isoprene produced into any other units. Exemplary equations are listed below for interconverting between units.

Units for Rate of Isoprene Production (Total and Specific) 1 g isoprene/L _(broth)/hr=14.7 mmol isoprene/L _(broth)/hr(total volumetric rate)  Equation 3 1 nmol isoprene/g _(wcm)/hr=1 nmol isoprene/L _(broth)/hr/OD₆₀₀ (This conversion assumes that one liter of broth with an OD₆₀₀ value of 1 has a wet cell weight of 1 gram.)  Equation 4 1 nmol isoprene/g _(wcm)/hr=68.1 ng isoprene/g _(wcm)/hr (given the molecular weight of isoprene)  Equation 5 1 nmol isoprene/L _(gas) O₂/hr=90 nmol isoprene/L _(broth)/hr (at an O₂ flow rate of 90 L/hr per L of culture broth)  Equation 6 1 μg isoprene/L _(gas) isoprene in off-gas=60 μg isoprene/L _(broth)/hr at a flow rate of 60 L _(gas) per L _(broth)(1 vvm)  Equation 7 Units for Titer (Total and Specific) 1 nmol isoprene/mg cell protein=150 nmol isoprene/L _(broth)/OD₆₀₀ (This conversion assumes that one liter of broth with an OD₆₀₀ value of 1 has a total cell protein of approximately 150 mg) (specific productivity)  Equation 8 1 g isoprene/L _(broth)=14.7 mmol isoprene/L _(broth)(total titer)  Equation 9

If desired, Equation 10 can be used to convert any of the units that include the wet weight of the cells into the corresponding units that include the dry weight of the cells. Dry weight of cells=(wet weight of cells)/3.3  Equation 10

If desired, Equation 11 can be used to convert between units of ppm and μg/L. In particular, “ppm” means parts per million defined in terms of μg/g (w/w). Concentrations of gases can also be expressed on a volumetric basis using “ppmv” (parts per million by volume), defined in terms of μL/L (vol/vol). Conversion of μg/L to ppm (e.g., μg of analyte per g of gas) can be performed by determining the mass per L of off-gas (i.e., the density of the gas). For example, a liter of air at standard temperature and pressure (STP; 101.3 kPa (1 bar) and 273.15K). has a density of approximately 1.29 g/L. Thus, a concentration of 1 ppm (μg/g) equals 1.29 μg/L at STP (equation 11). The conversion of ppm (μg/g) to μg/L is a function of both pressure, temperature, and overall composition of the off-gas. 1 ppm (μg/g) equals 1.29 μg/L at standard temperature and pressure (STP; 101.3 kPa (1 bar) and 273.15K).  Equation 11

Conversion of μg/L to ppmv (e.g., μL of analyte per L of gas) can be performed using the Universal Gas Law (equation 12). For example, an off-gas concentration of 1000 μg/L_(gas) corresponds to 14.7 μmol/L_(gas). The universal gas constant is 0.082057 L.atm K⁻¹mol⁻¹, so using equation 12, the volume occupied by 14.7 μmol of HG at STP is equal to 0.329 mL. Therefore, the concentration of 1000 μg/L HG is equal to 329 ppmv or 0.0329% (v/v) at STP. PV=nRT, where “P” is pressure, “V” is volume, “n” is moles of gas, “R” is the Universal gas constant, and “T” is temperature in Kelvin.  Equation 12

The amount of impurities in isoprene compositions are typically measured herein on a weight per volume (w/v) basis in units such as μg/L. If desired, measurements in units of μg/L can be converted to units of mg/m³ using equation 13. 1 μg/L=1 mg/m³  Equation 13

In some embodiments described herein, a cell comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide produces an amount of isoprene that is at least or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount of isoprene produced from a corresponding cell grown under essentially the same conditions without the heterologous nucleic acid encoding the isoprene synthase polypeptide.

In some embodiments described herein, a cell comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide and one or more heterologous nucleic acids encoding a DXS, IDI, and/or MVA pathway polypeptide produces an amount of isoprene that is at least or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount of isoprene produced from a corresponding cell grown under essentially the same conditions without the heterologous nucleic acids.

In some embodiments, the isoprene composition comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition has a relative detector response of greater than or about 99.90, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96, 99.97, 99.98, 99.99, or 100% for isoprene compared to the detector response for all C5 hydrocarbons in the composition. In some embodiments, the isoprene composition comprises between about 99.90 to about 99.92, about 99.92 to about 99.94, about 99.94 to about 99.96, about 99.96 to about 99.98, about 99.98 to 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.

In some embodiments, the isoprene composition comprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition has a relative detector response of less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for C5 hydrocarbons other than isoprene compared to the detector response for all C5 hydrocarbons in the composition. In some embodiments, the composition has a relative detector response of less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne compared to the detector response for all C5 hydrocarbons in the composition. In some embodiments, the isoprene composition comprises between about 0.02 to about 0.04%, about 0.04 to about 0.06%, about 0.06 to 0.08%, about 0.08 to 0.10%, or about 0.10 to about 0.12% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition.

In some embodiments, the isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene. In some embodiments, the isoprene composition comprises between about 0.005 to about 50, such as about 0.01 to about 10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005 μg/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene. In some embodiments, the isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a hydrocarbon other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne). In some embodiments, the isoprene composition comprises between about 0.005 to about 50, such as about 0.01 to about 10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005 lag/L of a hydrocarbon other than isoprene. In some embodiments, the isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a protein or fatty acid (such as a protein or fatty acid that is naturally associated with natural rubber).

In some embodiments, the isoprene composition comprises less than or about 10, 5, 1, 0.8, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of alpha acetylenes, piperylenes, acetonitrile, or 1,3-cyclopentadiene. In some embodiments, the isoprene composition comprises less than or about 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of sulfur or allenes. In some embodiments, the isoprene composition comprises less than or about 30, 20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of all acetylenes (such as 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne). In some embodiments, the isoprene composition comprises less than or about 2000, 1000, 500, 200, 100, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of isoprene dimers, such as cyclic isoprene dimmers (e.g., cyclic C10 compounds derived from the dimerization of two isoprene units).

In some embodiments, the isoprene composition includes ethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more of the foregoing. In particular embodiments, the isoprene composition comprises greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of ethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more of the foregoing. In some embodiments, the isoprene composition comprises between about 0.005 to about 120, such as about 0.01 to about 80, about 0.01 to about 60, about 0.01 to about 40, about 0.01 to about 30, about 0.01 to about 20, about 0.01 to about 10, about 0.1 to about 80, about 0.1 to about 60, about 0.1 to about 40, about 5 to about 80, about 5 to about 60, or about 5 to about 40 μg/L of ethanol, acetone, a C5 prenyl alcohol, or any two or more of the foregoing.

In some embodiments, the isoprene composition includes one or more of the following components: 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or a linear isoprene polymer (such as a linear isoprene dimer or a linear isoprene trimer derived from the polymerization of multiple isoprene units). In various embodiments, the amount of one of these components relative to amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w). In some embodiments, the relative detector response for the second compound compared to the detector response for isoprene is greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%. In various embodiments, the amount of one of these components relative to amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is between about 0.01 to about 105% (w/w), such as about 0.01 to about 90, about 0.01 to about 80, about 0.01 to about 50, about 0.01 to about 20, about 0.01 to about 10, about 0.02 to about 50, about 0.05 to about 50, about 0.1 to about 50, or 0.1 to about 20% (w/w).

In some embodiments, the isoprene composition includes one or more of the following: an alcohol, an aldehyde, or a ketone (such as any of the alcohols, aldehydes, or ketones described herein). In some embodiments, the isoprene composition includes (i) an alcohol and an aldehyde, (ii) an alcohol and a ketone, (iii) an aldehyde and a ketone, or (iv) an alcohol, an aldehyde, and a ketone.

In some embodiments, the isoprene composition contains one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid, 2-butanone, 2-methyl-1-butanol, or indole. In some embodiments, the isoprene composition contains 1 ppm or more of one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid, 2-butanone, 2-methyl-1-butanol, or indole. In some embodiments, the concentration of more of one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid, 2-butanone, 2-methyl-1-butanol, or indole, is between about 1 to about 10,000 ppm in an isoprene composition (such as off-gas before it is purified). In some embodiments, the isoprene composition (such as off-gas after it has undergone one or more purification steps) includes one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid, 2-butanone, 2-methyl-1-butanol, or indole, at a concentration between about 1 to about 100 ppm, such as about 1 to about 10 ppm, about 10 to about 20 ppm, about 20 to about 30 ppm, about 30 to about 40 ppm, about 40 to about 50 ppm, about 50 to about 60 ppm, about 60 to about 70 ppm, about 70 to about 80 ppm, about 80 to about 90 ppm, or about 90 to about 100 ppm. Volatile organic compounds from cell cultures (such as volatile organic compounds in the headspace of cell cultures) can be analyzed using standard methods such as those described herein or other standard methods such as proton transfer reaction-mass spectrometry (see, for example, Bunge et al., Applied and Environmental Microbiology, 74(7):2179-2186, 2008 which is hereby incorporated by reference in its entirety, particular with respect to the analysis of volatile organic compounds).

In some embodiments, the composition comprises greater than about 2 mg of isoprene, such as greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene. In some embodiments, the composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene. In some embodiments, the amount of isoprene in the composition is between about 2 to about 5,000 mg, such as between about 2 to about 100 mg, about 100 to about 500 mg, about 500 to about 1,000 mg, about 1,000 to about 2,000 mg, or about 2,000 to about 5,000 mg. In some embodiments, the amount of isoprene in the composition is between about 20 to about 5,000 mg, about 100 to about 5,000 mg, about 200 to about 2,000 mg, about 200 to about 1,000 mg, about 300 to about 1,000 mg, or about 400 to about 1,000 mg. In some embodiments, greater than or about 20, 25, 30, 40, 50, 60, 70, 80, 90, or 95% by weight of the volatile organic fraction of the composition is isoprene.

In some embodiments, the composition includes ethanol. In some embodiments, the composition includes between about 75 to about 90% by weight of ethanol, such as between about 75 to about 80%, about 80 to about 85%, or about 85 to about 90% by weight of ethanol. In some embodiments in which the composition includes ethanol, the composition also includes between about 4 to about 15% by weight of isoprene, such as between about 4 to about 8%, about 8 to about 12%, or about 12 to about 15% by weight of isoprene.

In some embodiments described herein, a cell comprising one or more heterologous nucleic acids encoding an isoprene synthase polypeptide, DXS polypeptide, IDI polypeptide, and/or MVA pathway polypeptide produces an amount of an isoprenoid compound (such as a compound with 10 or more carbon atoms that is formed from the reaction of one or more IPP molecules with one or more DMAPP molecules) that is greater than or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount of the isoprenoid compound produced from a corresponding cell grown under essentially the same conditions without the one or more heterologous nucleic acids. In some embodiments described herein, a cell comprising one or more heterologous nucleic acids encoding an isoprene synthase polypeptide, DXS polypeptide, IDI polypeptide, and/or MVA pathway polypeptide produces an amount of a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol) that is greater than or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount of the C5 prenyl alcohol produced from a corresponding cell grown under essentially the same conditions without the one or more heterologous nucleic acids.

Exemplary Co-Production of BioIsoprene and Hydrogen

In some embodiments, any of the isoprene-producing cells described herein that comprise one or more heterologous nucleic acids encoding an isoprene synthase polypeptide, a DXS polypeptide, an IDI polypeptide, and/or an MVA pathway polypeptide operably linked to a promoter further comprise a heterologous nucleic acid also operably linked to a promoter encoding one or more hydrogenase polypeptides or one or more polypeptides involved in the regulation or expression of hydrogenase polypeptides (e.g., hydrogenase maturation proteins or transcription factors). In some embodiments, any of the isoprene-producing cells described herein that comprise one or more heterologous nucleic acids encoding an isoprene synthase polypeptide, a DXS polypeptide, an IDI polypeptide, an MVA pathway polypeptide, one or more hydrogenase polypeptides or one or more polypeptides involved in the regulation or expression of hydrogenase polypeptides operably linked to a promoter further comprise a mutation or deletion inactivating one or more polypeptides involved in the production of fermentation side products, one or more polypeptides involved in the regulation or expression of genes for the production of fermentation side products, or one or more polypeptides involved in hydrogen reuptake. Such cells can co-produce isoprene and hydrogen.

In some embodiments of any of the aspects described herein, the cells are bacterial cells, such as gram-positive bacterial cells (e.g., Bacillus cells such as Bacillus subtilis cells or Streptomyces cells such as Streptomyces lividans, Streptomyces coelicolor, or Streptomyces griseus cells). In some embodiments of any of the aspects described herein, the cells are gram-negative bacterial cells (e.g., Escherichia cells such as Escherichia coli cells, Rhodopseudomonas sp. such as Rhodopseudomonas palustris cells, Pseudomonas sp. such as Pseudomonas fluorescens cells or Pseudomonas putida cells, or Pantoea cells such as Pantoea citrea cells). In some embodiments of any of the aspects described herein, the cells are fungal, cells such as filamentous fungal cells (e.g., Trichoderma cells such as Trichoderma reesei cells or Aspergillus cells such as Aspergillus oryzae and Aspergillus niger) or yeast cells (e.g., Yarrowia cells such as Yarrowia lipolytica cells or Saccharomyces cells such as Saccharomyces cerevisiae).

In some embodiments of any of the aspects described herein, the isoprene synthase polypeptide is a polypeptide from a plant such as Pueraria (e.g., Pueraria montana or Pueraria lobata) or Populus (e.g., Populus tremuloides, Populus alba, Populus nigra, Populus trichocarpa, or the hybrid, Populus alba x Populus tremula).

In some embodiments of any of the aspects described herein, the cells further comprise a heterologous nucleic acid encoding an IDI polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding an IDI polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise a heterologous nucleic acid encoding a DXS polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding a DXS polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise one or more nucleic acids encoding an IDI polypeptide and a DXS polypeptide. In some embodiments of any of the aspects described herein, one nucleic acid encodes the isoprene synthase polypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments of any of the aspects described herein, one vector encodes the isoprene synthase polypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments, the vector comprises a selective marker or a selectable marker, such as an antibiotic resistance nucleic acid.

In some embodiments of any of the aspects described herein, the cells further comprise a heterologous nucleic acid encoding an MVA pathway polypeptide (such as an MVA pathway polypeptide from Saccharomyces cerevisia or Enterococcus faecalis). In some embodiments of any of the aspects described herein, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding an MVA pathway polypeptide (such as an MVA pathway polypeptide from Saccharomyces cerevisia or Enterococcus faecalis). In some embodiments of any of the aspects described herein, the cells comprise an isoprene synthase, DXS, and MVA pathway nucleic acid. In some embodiments of any of the aspects described herein, the cells comprise an isoprene synthase nucleic acid, a DXS nucleic acid, an IDI nucleic acid, and a MVA pathway nucleic acid.

In some embodiments, the isoprene-producing cells described herein further comprise a heterologous nucleic acid encoding a hydrogenase polypeptide operably linked to a promoter. In some embodiments, the hydrogenase polypeptide comprises E. coli hydrogenase-1 (Hyd-1), E. coli hydrogenase-2 (Hyd-2), E. coli hydrogenase-3 (Hyd-3), E. coli hydrogenase-4 (Hyd-4), E. coli formate hydrogen lyase (FHL) complex, which produces hydrogen gas from formate and CO₂ under anaerobic conditions at acidic pH, Rhodococcus opacus MR11 hydrogenase (R. opacus HoxH), Synechosystis sp. PCC 6803 hydrogenase (Syn. PCC 6803 HoxH), Desulfovibrio gigas hydrogenase (D. gigas), and Desulfovibrio desulfuricans ATCC 7757 hydrogenase (D. desulfuricans). In some embodiments, the isoprene-producing cells further comprising a heterologous nucleic acid encoding a hydrogenase polypeptide operably linked to a promoter further comprise E. coli hydrogenase-3 (Hyd-3), E. coli pyruvate formate lyase (pfl), and E. coli formate hydrogen lyase (FHL) complex.

In some embodiments, the hydrogenase polypeptide encodes a ferredoxin-dependent hydrogenase polypeptide. In some embodiments, the ferredoxin-dependent hydrogenase polypeptide comprises Clostridium acetobutulicum hydrogenase A (HydA), which can be expressed in conjunction with one or more of: (1) Bacillus subtilis NADPH ferredoxin oxidoreductase (NFOR) or Clostridium kluyveri NADH ferredoxin oxidoreductase (RnfCDGEAB), Clostridium pasteuranium ferredoxin oxidoreductase (Fdx); (2) glyceraldehyde-6-phosphate ferredoxin oxidoreductase (GAPOR); or (3) pyruvate ferredoxin oxidoreductase (POR). In some embodiments, the ferredoxin-dependent hydrogenase polypeptide Clostridium acetobutulicum hydrogenase A (HydA) is expressed with three HydA-associated maturation enzymes (HydE, HydG, and HydF), and further in conjunction with one or more of: (1) Bacillus subtilis NADPH ferredoxin oxidoreductase (NFOR) or Clostridium kluyveri NADH ferredoxin oxidoreductase (RnfCDGEAB), Clostridium pasteuranium ferredoxin oxidoreductase (Fdx); (2) glyceraldehyde-6-phosphate ferredoxin oxidoreductase (GAPOR); or (3) pyruvate ferredoxin oxidoreductase (POR).

In some embodiments, the hydrogenase polypeptide encodes an NADPH-dependent hydrogenase polypeptide. In some embodiments, the NADPH-dependent hydrogenase polypeptide comprises Pyrococcus furiosus hydrogenase. In some embodiments, the hydrogenase polypeptide encodes an oxygen-tolerant hydrogenase. In some embodiments, the oxygen-tolerant hydrogenase comprises Rubrivivax gelatinosus hydrogenase, and Ralstonia eutropha hydrogenase.

In some embodiments, the isoprene-producing cells described herein further comprise a mutation or deletion inactivating a gene involved in regulation of hydrogenase activity, such as iron-sulfur complex transcriptional regulator (iscR) (Kalim-Akhtar et al., “Deletion of iscR stimulates recombinant Clostridial Fe/Fe hydrogenase activity and H₂-accumulation in Escherichia coli BL21(DE3),” Appl. Microbiol. Biotechnol. 78:853-862 (2008), which is incorporated herein by reference in its entirety, particularly with reference to stimulation of Clostridial Fe/Fe hydrogenase activity and hydrogen accumulation in E. coli by deleting the iscR gene).

In some embodiments, the isoprene-producing cells described herein further comprise a mutation or deletion inactivating a gene encoding one or more cellular polypeptides involved in production of fermentation side products, such as lactate, acetate, pyruvate, ethanol, succinate, and glycerol. In some embodiments, the inactivated polypeptides involved in production of fermentation side products comprise one or more polypeptides encoding formate dehydrogenase N, alpha subunit (fdnG), formate dehydrogenase O, large subunit (fdoG), nitrate reductase (narG), formate transporter A (focA), formate transporter B (focB), pyruvate oxidase (poxB), pyruvate dehydrogenase E1 component ackA/pta (aceE), alcohol dehydrogenase (adhE), fumarate reductase membrane protein (frdC), or lactate dehydrogenase (ldhA).

In some embodiments, the isoprene-producing cells described herein further comprise a mutation or deletion inactivating a gene encoding one or more cellular polypeptides involved in the regulation or expression of genes involved in production of fermentation side products. In some embodiments, the inactivated polypeptides involved in the regulation or expression of genes involved in production of fermentation side products comprise repressor of formate hydrogen lyase (hycA), fumarate reductase regulator (fnr), acetyl-coenzyme A synthetase (acs), and formate dehydrogenase regulatory protein (hycA).

In some embodiments, the isoprene-producing cells described herein further comprise a mutation or deletion inactivating a gene encoding one or more cellular polypeptides involved in hydrogen re-uptake. In some embodiments, the inactivated polypeptides involved in hydrogen re-uptake comprise E. coli hydrogenase-1 (Hyd-1) (hya operon) and E. coli hydrogenase-2 (Hyd-2) (hyb operon).

In some embodiments of any of the aspects described herein, the heterologous isoprene synthase, DXS polypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptide or nucleic acid is operably linked to a T7 promoter, such as a T7 promoter contained in a medium or high copy plasmid. In some embodiments of any of the aspects described herein, the heterologous isoprene synthase, DXS polypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid is operably linked to a Trc promoter, such as a Trc promoter contained in a medium or high copy plasmid. In some embodiments of any of the aspects described herein, the heterologous isoprene synthase, DXS polypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid is operably linked to a Lac promoter, such as a Lac promoter contained in a low copy plasmid. In some embodiments of any of the aspects described herein, the heterologous isoprene synthase, DXS polypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid is operably linked to an endogenous promoter, such as an endogenous alkaline serine protease promoter. In some embodiments, the heterologous isoprene synthase, DXS polypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid integrates into a chromosome of the cells without a selective marker or without a selectable marker.

In some embodiments, one or more MVA pathway, IDI, DXS, isoprene synthase, hydrogenase, hydrogenase maturation or transcription factor nucleic acids are placed under the control of a promoter or factor that is more active in stationary phase than in the growth phase. For example, one or more MVA pathway, IDI, DXS, isoprene synthase, hydrogenase, hydrogenase maturation or transcription factor nucleic acids may be placed under control of a stationary phase sigma factor, such as RpoS. In some embodiments, one or more MVA pathway, IDI, DXS, isoprene synthase, hydrogenase, hydrogenase maturation or transcription factor nucleic acids are placed under control of a promoter inducible in stationary phase, such as a promoter inducible by a response regulator active in stationary phase.

In some embodiments of any of the aspects described herein, at least a portion of the cells maintain the heterologous isoprene synthase, DXS polypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments of any of the aspects described herein, the nucleic acid comprising the isoprene synthase, DXS polypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid also comprises a selective marker or a selectable marker, such as an antibiotic resistance nucleic acid.

In some embodiments of any of the aspects described herein, cells that co-produce isoprene and hydrogen are cultured in any of the culture media described herein, under oxygen-limited conditions to facilitate the co-production of isoprene and hydrogen by the cells. In some embodiments, the cells are grown in oxygen-limited culture. In some embodiments, the cells are grown in the presence of 0.5 moles of oxygen per mole of isoprene. In some embodiments, the cells are grown anaerobically, in the absence of oxygen.

In some embodiments, any of the cells described herein are grown in oxygen-limited culture and co-produce isoprene and hydrogen. In some embodiments, the cells in oxygen-limited culture produce isoprene at a rate greater than about 400 nmole/g_(wcm)/hr, and produce hydrogen at a rate greater than about 125 nmole/g_(wcm)/hr. In some embodiments, the cells in oxygen-limited culture produce isoprene at a rate between about 400 nmole/g_(wcm)/hr to about 2.0×10⁵ nmole/g_(wcm)/hr and hydrogen at a rate between about 125 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr. In some embodiments, the cells in oxygen-limited culture produce isoprene at a rate between about 400 nmole/g_(wcm)/hr and about 2.0×10⁵ nmole/g_(wcm)/hr, between about 500 nmole/g_(wcm)/hr and about 1.5×10⁵ nmole/g_(wcm)/hr, between about 750 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, between about 1000 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, between about 2500 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, between about 5000 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, between about 7500 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, and between about 1×10⁴ nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr. In some embodiments, the cells in oxygen-limited culture produce greater than about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/g_(wcm)/hr isoprene. In some embodiments, the cells in oxygen-limited culture produce hydrogen at a rate between about 125 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 250 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 750 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 1000 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 1250 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 2500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 5000 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 7500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, and between about 1.00×10⁴ nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr. In some embodiments, the cells in oxygen-limited culture produce greater than about 125, 250, 500, 750, 1000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, 10,000, or more nmole/g_(wcm)/hr hydrogen.

In some embodiments, any of the cells described herein are grown in oxygen-limited culture and co-produce isoprene and hydrogen. In some embodiments, the cells in oxygen-limited culture have an average volumetric productivity of isoprene greater than about 0.1 mg/L_(broth)/hr and an average volumetric productivity of hydrogen greater than about 0.005 mg/L_(broth)/hr. In some embodiments, the cells in oxygen-limited culture have a peak volumetric productivity of isoprene greater than about 1000 mg/L_(broth)/hr and a peak volumetric productivity of hydrogen greater than about 5 mg/L_(broth)/hr. In some embodiments, the cells in oxygen-limited culture have a peak volumetric productivity of isoprene greater than about 3000 mg/L_(broth)/hr and a peak volumetric productivity of hydrogen greater than about 5 mg/L_(broth)/hr. In some embodiments, the cells in oxygen-limited culture have a peak volumetric productivity of isoprene greater than about 5000 mg/L_(broth)/hr and a peak volumetric productivity of hydrogen greater than about 5 mg/L_(broth)/hr. In some embodiments, the cells in oxygen-limited culture have an average volumetric productivity of isoprene between about 0.1 mg/L_(broth)/hr and about 5000 mg/L_(broth)/hr, and an average volumetric productivity of hydrogen between about 0.005 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr. In some embodiments, the cells in oxygen-limited culture have an average volumetric productivity of isoprene between about 1 mg/L_(broth)/hr and about 5000 mg/L_(broth)/hr, between about 5 mg/L_(broth)/hr and about 5000 mg/L_(broth)/hr, between about 10 mg/L_(broth)/hr and about 5000 mg/L_(broth)/hr, between about 25 mg/L_(broth)/hr and about 5000 mg/L_(broth)/hr, between about 50 mg/L_(broth)/hr and about 5000 mg/L_(broth)/hr, between about 100 mg/L_(broth)/hr and about 5000 mg/L_(broth)/hr, between about 250 mg/L_(broth)/hr and about 5000 mg/L_(broth)/hr, between about 500 mg/L_(broth)/hr and about 5000 mg/L_(broth)/hr, between about 1000 mg/L_(broth)/hr and about 5000 mg/L_(broth)/hr, and between about 2500 mg/L_(broth)/hr and about 5000 mg/L_(broth)/hr, and an average volumetric productivity of hydrogen between about 0.01 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, between about 0.025 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, between about 0.05 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, between about 0.1 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, between about 0.25 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, between about 0.5 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, between about 1 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, and between about 2.5 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr.

In some embodiments, any of the cells described herein are grown in oxygen-limited culture and co-produce isoprene and hydrogen. In some embodiments, the cells in oxygen-limited culture convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, and produce hydrogen equivalent to more than about 0.024 molar percent of the carbon that the cells consume from a cell culture medium. In some embodiments, the cells in oxygen-limited culture convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, and produce hydrogen equivalent to more than about 400 molar percent of the carbon that the cells consumer from a cell culture medium.

In some embodiments, any of the cells described herein that co-produce isoprene and hydrogen are grown in oxygen-limited culture. In some embodiments, the cells in oxygen-limited culture co-produce isoprene and hydrogen in a ratio ranging from at least one molar percent of isoprene for every three molar percent of hydrogen to at least one molar percent of isoprene for every four molar percent of hydrogen. In some embodiments, the cells in oxygen-limited culture produce an off-gas containing from 1 to 11 molar percent isoprene and from 3 to 33 molar percent hydrogen. In some embodiments, the cells produce from 1 to 11 molar percent isoprene and from 4 to 44 molar percent hydrogen. In some embodiments, the cells in oxygen-limited culture also produce an off-gas containing oxygen, carbon dioxide, or nitrogen. In some embodiments, the cells in oxygen limited culture produce an off-gas containing from 0 to 21 molar percent oxygen, from 18 to 44 molar percent carbon dioxide, and from 0 to 78 molar percent nitrogen.

In another aspect, provided herein are cells in oxygen-limited culture that co-produce isoprene and hydrogen, comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the cells: (i) produce isoprene at a rate greater than about 400 nmole/g_(wcm)/hr and produce hydrogen at a rate greater than about 125 nmole/g_(wcm)/hr; (ii) have an average volumetric productivity of isoprene greater than about 0.1 mg/L_(broth)/hr and an average volumetric productivity of hydrogen greater than about 0.005 mg/L_(broth)/hr; or (iii) convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, and produce hydrogen equivalent to more than about 0.024 molar percent of the carbon that the cells consume from a cell culture medium.

In some embodiments, the cells in oxygen-limited culture comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter, and wherein the cells produce greater than about 400 nmole/g_(wcm)/hr of isoprene and greater than about 125 nmole/g_(wcm)/hr of hydrogen. In some embodiments, the cells in oxygen-limited culture comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter, and wherein the cells have an average volumetric productivity of isoprene greater than about 0.1 mg/L_(broth)/hr and an average volumetric productivity of hydrogen greater than about 0.005 mg/L_(broth)/hr. In some embodiments, the cells in oxygen-limited culture comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter, and wherein the cells convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, and more than about 0.024 molar percent of the carbon that the cells consume from a cell culture medium into hydrogen. In some embodiments, the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

In some embodiments, the cells in oxygen-limited culture comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide produce isoprene at a rate between about 400 nmole/g_(wcm)/hr and about 2.0×10⁵ nmole/g_(wcm)/hr, between about 500 nmole/g_(wcm)/hr and about 1.5×10⁵ nmole/g_(wcm)/hr, between about 750 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, between about 1000 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, between about 2500 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, between about 5000 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, between about 7500 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, and between about 1×10⁴ nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, and produce hydrogen at a rate between about 125 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 250 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 750 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 1000 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 1250 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 2500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 5000 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 7500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, and between about 1.00×10⁴ nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr.

In some embodiments, provided herein are methods of co-producing isoprene and hydrogen, the methods comprising: (a) culturing cells under conditions suitable for the co-production of isoprene and hydrogen; and (b) co-producing isoprene and hydrogen, wherein the cells produce greater than about 400 nmole/g_(wcm)/hour of isoprene, and wherein the cells produce greater than about 125 nmole/g_(wcm)/hr of hydrogen.

In some embodiments, the cells in oxygen-limited culture comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide produce isoprene at a rate between about 400 nmole/g_(wcm)/hr and about 2.0×10⁵ nmole/g_(wcm)/hr, between about 500 nmole/g_(wcm)/hr and about 1.5×10⁵ nmole/g_(wcm)/hr, between about 750 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, between about 1000 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, between about 2500 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, between about 5000 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, between about 7500 nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, and between about 1×10⁴ nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr, and produce hydrogen at a rate between about 125 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 250 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 750 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 1000 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 1250 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 2500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 5000 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, between about 7500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, and between about 1.00×10⁴ nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr.

In some embodiments, provided herein are methods of co-producing isoprene and hydrogen, the methods comprising: (a) culturing cells under conditions suitable for the co-production of isoprene and hydrogen; and (b) co-producing isoprene and hydrogen, wherein the cells have an average volumetric productivity of isoprene greater than about 0.1 mg/L_(broth)/hr and an average volumetric productivity of hydrogen greater than about 0.005 mg/L_(broth)/hr.

In some embodiments, provided herein are methods of co-producing isoprene and hydrogen, the methods comprising: (a) culturing cells under conditions suitable for the co-production of isoprene and hydrogen; and (b) co-producing isoprene and hydrogen, wherein the cells convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, and produce hydrogen equivalent to more than about 0.024 molar percent of the carbon that the cells consume from a cell culture medium.

In some embodiments, provided herein are compositions comprising isoprene and hydrogen in a ratio ranging from at least one molar percent of isoprene for every three molar percent of hydrogen to at least one molar percent of isoprene for every four molar percent of hydrogen, and 0.1 molar percent or less of volatile impurities. In some embodiments, the compositions further comprise from 1 to 11 molar percent isoprene and from 4 to 44 molar percent hydrogen. In some embodiments, the compositions further comprise oxygen, carbon dioxide, or nitrogen. In some embodiments, the compositions further comprise from 0 to 21 molar percent oxygen, from 18 to 44 molar percent carbon dioxide, and from 0 to 78 molar percent nitrogen. In some embodiments, the composition further comprises 1.0×10⁻⁴ molar percent or less of non-methane volatile impurities. In some embodiments, the non-methane volatile impurities comprise one or more of the following: 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or a linear isoprene polymer (such as a linear isoprene dimer or a linear isoprene trimer derived from the polymerization of multiple isoprene units). In some embodiments, the non-methane volatile impurities comprise one or more of the following: the isoprene composition includes one or more of the following: an alcohol, an aldehyde, or a ketone (such as any of the alcohols, aldehydes, or ketones described herein). In some embodiments, the isoprene composition includes (i) an alcohol and an aldehyde, (ii) an alcohol and a ketone, (iii) an aldehyde and a ketone, or (iv) an alcohol, an aldehyde, and a ketone. In some embodiments, the non-methane volatile impurities comprise one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid, 2-butanone, 2-methyl-1-butanol, or indole.

Also provided herein are methods of co-producing isoprene and hydrogen, the methods comprising: a) culturing cells under conditions suitable for the co-production of isoprene and hydrogen; and b) co-producing isoprene and hydrogen, wherein the peak concentration of the isoprene produced by the cells in oxygen-limited culture is greater than about 10 ng/L_(broth) and the hydrogen evolution rate of the cells is greater than about 0.0025 mmol/L_(broth)/hour. In some embodiments of any of these methods, the hydrogen evolution rate is between about any of 0.0025 mmol/L_(broth)/hr and about 10 mmol/L_(broth)/hr, between about 0.0025 mmol/L_(broth)/hr and about 5 mmol/L_(broth)/hr, between about 0.0025 mmol/L_(broth)/hr and about 2.5 mmol/L_(broth)/hr, between about 0.0025 mmol/L_(broth)/hr and about 1 mmol/L_(broth)/hr, between about 0.0025 mmol/L_(broth)/hr and about 0.5 mmol/L_(broth)/hr, between about 0.0025 mmol/L_(broth)/hr and about 0.25 mmol/L_(broth)/hr, between about 0.0025 mmol/L_(broth)/hr and about 0.025 mmol/L_(broth)/hr, between about 0.025 mmol/L_(broth)/hr and about 0.5 mmol/L_(broth)/hr, between about 0.025 mmol/L_(broth)/hr and about 1 mmol/L_(broth)/hr, between about 0.025 mmol/L_(broth)/hr and about 2.5 mmol/L_(broth)/hr, between about 0.025 mmol/L_(broth)/hr and about 5 mmol/L_(broth)/hr, between about 0.025 mmol/L_(broth)/hr and about 10 mmol/L_(broth)/hr, between about 0.25 mmol/L_(broth)/hr and 1 mmol/L_(broth)/hr, between about 0.25 mmol/L_(broth)/hr and 2.5 mmol/L_(broth)/hr, between about 0.25 mmol/L_(broth)/hr and 2.5 mmol/L_(broth)/hr, and between about 0.25 mmol/L_(broth)/hr and 10 mmol/L_(broth)/hr.

Provided herein are also methods of co-producing isoprene and hydrogen comprising a) culturing cells under conditions suitable for the co-production of isoprene and hydrogen; and b) co-producing isoprene and hydrogen, wherein the liquid phase concentration of isoprene is less than about 200 mg/L, the cells produce greater than about 400 nmole/g_(wcm)/hour of isoprene, and the hydrogen evolution rate of the cells is greater than about 0.0025 mmol/L/hour. In some embodiments, the liquid phase concentration of isoprene in the culture is less than about any of 175 mg/L, 150 mg/L, 125 mg/L, 100 mg/L, 75 mg/L, 50 mg/L, 25 mg/L, 20 mg/L, 15 mg/L, 10 mg/L, 5 mg/L, or 2.5 mg/L. In some embodiments, the liquid phase concentration of isoprene in culture is between about any of 0.1 mg/L to 200 mg/L, 1 mg/L to 200 mg/L, 1 mg/L to 150 mg/L, 1 mg/L to 100 mg/L, 1 mg/L to 50 mg/L, 1 mg/L to 25 mg/L, 1 mg/L to 20 mg/L, or 10 mg/L to 20 mg/L. In some embodiments of any of these methods, the hydrogen evolution rate is between about any of 0.0025 mmol/L_(broth)/hr and about 10 mmol/L_(broth)/hr, between about 0.0025 mmol/L_(broth)/hr and about 5 mmol/L_(broth)/hr, between about 0.0025 mmol/L_(broth)/hr and about 2.5 mmol/L_(broth)/hr, between about 0.0025 mmol/L_(broth)/hr and about 1 mmol/L_(broth)/hr, between about 0.0025 mmol/L_(broth)/hr and about 0.5 mmol/L_(broth)/hr, between about 0.0025 mmol/L_(broth)/hr and about 0.25 mmol/L_(broth)/hr, between about 0.0025 mmol/L_(broth)/hr and about 0.025 mmol/L_(broth)/hr, between about 0.025 mmol/L_(broth)/hr and about 0.5 mmol/L_(broth)/hr, between about 0.025 mmol/L_(broth)/hr and about 1 mmol/L_(broth)/hr, between about 0.025 mmol/L_(broth)/hr and about 2.5 mmol/L_(broth)/hr, between about 0.025 mmol/L_(broth)/hr and about 5 mmol/L_(broth)/hr, between about 0.025 mmol/L_(broth)/hr and about 10 mmol/L_(broth)/hr, between about 0.25 mmol/L_(broth)/hr and 1 mmol/L_(broth)/hr, between about 0.25 mmol/L_(broth)/hr and 2.5 mmol/L_(broth)/hr, between about 0.25 mmol/L_(broth)/hr and 2.5 mmol/L_(broth)/hr, and between about 0.25 mmol/L_(broth)/hr and 10 mmol/L_(broth)/hr.

In one aspect, provided herein are cells in oxygen-limited culture that co-produce isoprene and hydrogen. In some embodiments, the oxygen-limited culture is anaerobic. In some embodiments, the cells in oxygen-limited culture produce greater than about 400 nmole/g_(wcm)/hr of isoprene and greater than about 125 nmole/g_(wcm)/hr of hydrogen. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

In some embodiments, provided herein are cells in oxygen-limited culture that convert more than about 0.002% of the carbon in a cell culture medium into isoprene and produce hydrogen equivalent to more than about 0.024 molar percent of the carbon in a cell culture medium. In some embodiments, the oxygen-limited culture is anaerobic. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

In some embodiments, provided herein are cells in oxygen-limited culture that comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide. In some embodiments, the oxygen-limited culture is anaerobic. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

In one aspect, provided herein are methods of co-producing isoprene with another compound, such as methods of using any of the cells described herein to co-produce isoprene and hydrogen. In some embodiments, the method involves culturing cells under oxygen-limited conditions sufficient to produce greater than about 400 nmole/g_(wcm)/hr of isoprene and greater than about 125 nmole/g_(wcm)/hr of hydrogen. In some embodiments, the oxygen-limited culture is anaerobic. In some embodiments, the method also includes recovering the isoprene and hydrogen produced by the cells. In some embodiments, the method further includes purifying the isoprene and the hydrogen produced by the cells. In some embodiments, the method includes polymerizing the isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions. In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD₆₀₀) during stationary phase is greater than or about 2 or more times the amount of isoprene produced during the growth phase for the same length of time.

In some embodiments, the method includes culturing cells under oxygen-limited conditions sufficient to convert more than about 0.002% of the carbon (mol/mol) in a cell culture medium into isoprene and to produce hydrogen equivalent to more than about 0.024 molar percent of the carbon in a cell culture medium. In some embodiments, the oxygen-limited culture is anaerobic. In some embodiments, the method also includes recovering isoprene and hydrogen produced by the cells. In some embodiments, the method further includes purifying isoprene and hydrogen produced by the cells. In some embodiments, the method includes polymerizing the isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing.

In some embodiments of any of the aspects described herein, the microbial polypeptide carbon source includes one or more polypeptides from yeast or bacteria. In some embodiments of any of the aspects described herein, the plant polypeptide carbon source includes one or more polypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.

In some embodiments, isoprene and hydrogen are only co-produced in stationary phase. In some embodiments, isoprene and hydrogen are co-produced in both the growth phase and stationary phase. In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD₆₀₀) during stationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amount of isoprene produced during the growth phase for the same length of time. In various embodiments, the amount of hydrogen produced (such as the total amount of hydrogen produced or the amount of hydrogen produced per liter of broth per hour per OD₆₀₀) during stationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amount of hydrogen produced during the growth phase for the same length of time.

In some embodiments, the compositions provided herein comprise hydrogen and greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition comprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition has less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared to the total weight of all C5 hydrocarbons in the composition. In particular embodiments, the composition has greater than about 2 mg of isoprene and has greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene. In particular embodiments, the composition also comprises greater than about 2 mg of isoprene and greater than about 0.48 mg of hydrogen.

In some embodiments, the volatile organic fraction of the gas phase has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a compound that inhibits the polymerization of isoprene for any compound in the volatile organic fraction of the gas phase that inhibits the polymerization of isoprene. In particular embodiments, the volatile organic fraction of the gas phase also has greater than about 2 mg of isoprene and greater than about 0.48 mg of hydrogen.

In some embodiments, the systems include any of the cells and/or compositions described herein. In some embodiments, the system includes a reactor that chamber comprises cells in oxygen-limited culture that produce greater than about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/g_(wcm)/hr isoprene and greater than about 125, 250, 500, 750, 1000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, 10,000, or more nmole/g_(wcm)/hr hydrogen. In some embodiments, the system is not a closed system. In some embodiments, at least a portion of the isoprene is removed from the system. In some embodiments, the system includes a gas phase comprising isoprene and hydrogen. In various embodiments, the gas phase comprises any of the compositions described herein.

In one aspect, featured herein is a product produced by any of the compositions or methods described herein.

Cell Viability at High Isoprene Titer

Isoprene is a hydrophobic molecule secreted by many plants, animals, and microbes. Bacteria, such as Bacillus, produce isoprene at fairly low levels. While there is some evidence that plants secrete isoprene to help with thermoprotection, it has been hypothesized that isoprene may act antagonistically to cyanobacteria or fungi, or as an antimicrobial agent. See, e.g., Ladygina et al., Process Biochemistry 41:1001-1014 (2006), which is incorporated by reference in its entirety, particularly with respect to isoprene acting antagonistically. Since the very low production levels happening in nature are sufficient to be anti-microbial, it was of great concern that the titers and productivity levels of isoprene necessary for commercialization of isoprene would kill the host microbe.

We have found methods for producing titers and productivity levels of isoprene for commercialization of isoprene while maintaining cell viability and/or metabolic activity as indicated by carbon dioxide evolution rate or total carbon dioxide evolution rate.

Provided herein are methods of producing isoprene comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein cells produce greater than about 400 nmole/g_(wcm)/hour of isoprene, and the carbon dioxide evolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In some embodiments, the isoprene produced is any concentration or amount disclosed in the section entitled “Exemplary Production of Isoprene.” In some embodiments, the amount of isoprene is between about any of 400 nmole/g_(wcm)/hour to 1 mole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 1 mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 40 mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 4 mmole/g_(wcm)/hour, 1 mmole/g_(wcm)/hour to 1.5 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour to 3 mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour to 5 mmole/g_(wcm)/hour, 5 mmole/g_(wcm)/hour to 25 mmole/g_(wcm)/hour, 25 mmole/g_(wcm)/hour to 100 mmole/g_(wcm)/hour, 100 mmole/g_(wcm)/hour to 500 mmole/g_(wcm)/hour, or 500 mmole/g_(wcm)/hour to 1000 mmole/g_(wcm)/hour. In some embodiments, the amount of isoprene is about any of 1 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour, 2 mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour, 4 mmole/g_(wcm)/hour, or 5 mmole/g_(wcm)/hour. In some embodiments, the carbon dioxide evolution rate is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

Provided herein are also methods of producing isoprene comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein cells produce greater than about 400 nmole/g_(wcm)/hour of isoprene, and cell viability is reduced by less than about two-fold. In some embodiments, the isoprene produced is any concentration or amount disclosed in the section entitled “Exemplary Production of Isoprene.” In some embodiments, the amount of isoprene is between about any of 400 nmole/g_(wcm)/hour to 1 mole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 1 mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 40 mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 4 mmole/g_(wcm)/hour, 1 mmole/g_(wcm)/hour to 1.5 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour to 3 mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour to 5 mmole/g_(wcm)/hour, 5 mmole/g_(wcm)/hour to 25 mmole/g_(wcm)/hour, 25 mmole/g_(wcm)/hour to 100 mmole/g_(wcm)/hour, 100 mmole/g_(wcm)/hour to 500 mmole/g_(wcm)/hour, or 500 mmole/g_(wcm)/hour to 1000 mmole/g_(wcm)/hour. In some embodiments, the amount of isoprene is about any of 1 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour, 2 mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour, 4 mmole/g_(wcm)/hour, or 5 mmole/g_(wcm)/hour. In some embodiments, cell viability is reduced by less than about any of 1.75-fold, 1.5-fold, 1.25-fold, 1-fold, 0.75-fold, 0.5-fold, or 0.25-fold. In some embodiments, cell viability is reduced by about 2-fold.

Further provided herein are methods of producing isoprene comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the cumulative total productivity of the isoprene produced by the cells in culture is greater than about 0.2 mg/L_(broth)/hour and the carbon dioxide evolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In some embodiments, the cumulative total productivity of isoprene is any concentration or amount disclosed in the section entitled “Exemplary Production of Isoprene.” In some embodiments, the cumulative total productivity of the isoprene is between about any of 0.2 mg/L_(broth)/hour to 5 g/L_(broth)/hour, 0.2 mg/L_(broth)/hour to 1 g/L_(broth)/hour, 1 g/L_(broth)/hour to 2.5 g/L_(broth)/hour, 2.5 g/L_(broth)/hour to 5 g/L_(broth)/hour. In some embodiments, the carbon dioxide evolution rate is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

Provided herein are methods of producing isoprene comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the cumulative total productivity of the isoprene produced by the cells in culture is greater than about 0.2 mg/L_(broth)/hour and cell viability is reduced by less than about two-fold. In some embodiments, the cumulative total productivity of isoprene is any concentration or amount disclosed in the section entitled “Exemplary Production of Isoprene.” In some embodiments, the cumulative total productivity of the isoprene is between about any of 0.2 mg/L_(broth)/hour to 5 g/L_(broth)/hour, 0.2 mg/L_(broth)/hour to 1 g/L_(broth)/hour, 1 g/L_(broth)/hour to 2.5 g/L_(broth)/hour, 2.5 g/L_(broth)/hour to 5 g/L_(broth)/hour. In some embodiments, cell viability is reduced by less than about any of 1.75-fold, 1.5-fold, 1.25-fold, 1-fold, 0.75-fold, 0.5-fold, or 0.25-fold.

Methods of producing isoprene are also provided herein comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the peak concentration of the isoprene produced by the cells in culture is greater than about 10 ng/L_(broth) and the carbon dioxide evolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In some embodiments, the peak concentration of isoprene is any concentration or amount disclosed in the section entitled “Exemplary Production of Isoprene.” In some embodiments, the peak concentration of isoprene is between about any of 10 ng/L_(broth) to 500 ng/L_(broth), 500 ng/L_(broth) to 1 μg/L_(broth), 1 μg/L_(broth) to 5 μg/L_(broth), 5 μg/L_(broth) to 50 μg/L_(broth), 5 μg/L_(broth) to 100 μg/L_(broth), 5 μg/L_(broth) to 250 μg/L_(broth), 250 μg/L_(broth) to 500 μg/L_(broth), 500 μg/L_(broth) to 1 mg/L_(broth), 1 mg/L_(broth) to 50 mg/L_(broth), 1 mg/L_(broth) to 100 mg/L_(broth), 1 mg/L_(broth) to 200 mg/L_(broth), 10 ng/L_(broth) to 200 mg/L_(broth), 5 μg/L_(broth) to 100 mg/L_(broth), or 5 μg/L_(broth) to 200 mg/L_(broth). In some embodiments, the peak concentration is any of about 10 ng/L_(broth), 100 ng/L_(broth), 1 μg/L_(broth), 5 μg/L_(broth), 1 mg/L_(broth), 30 mg/L_(broth), 100 mg/L_(broth), or 200 mg/L_(broth). In some embodiments, the carbon dioxide evolution rate is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

In addition, methods of producing isoprene are also provided herein comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the peak concentration of the isoprene produced by the cells in culture is greater than about 10 ng/L_(broth) and cell viability is reduced by less than about two-fold. In some embodiments, the peak concentration of isoprene is any concentration or amount disclosed in the section entitled “Exemplary Production of Isoprene.” In some embodiments, the peak concentration of isoprene is between about any of 10 ng/L_(broth) to 500 ng/L_(broth), 500 ng/L_(broth) to 1 μg/L_(broth), 1 μg/L_(broth) to 5 μg/L_(broth), 5 μg/L_(broth) to 50 μg/L_(broth), 5 μg/L_(broth) to 100 μg/L_(broth), 5 μg/L_(broth) to 250 μg/L_(broth), 250 μg/L_(broth) to 500 μg/L_(broth), 500 μg/L_(broth) to 1 mg/L_(broth), 1 mg/L_(broth) to 50 mg/L_(broth), 1 mg/L_(broth) to 100 mg/L_(broth), 1 mg/L_(broth) to 200 mg/L_(broth), 10 ng/L_(broth) to 200 mg/L_(broth), 5 μg/L_(broth) to 100 mg/L_(broth), or 5 μg/L_(broth) to 200 mg/L_(broth). In some embodiments, the peak concentration is any of about 10 ng/L_(broth), 100 ng/L_(broth), 1 μg/L_(broth), 5 μg/L_(broth), 1 mg/L_(broth), 30 mg/L_(broth), 100 mg/L_(broth), or 200 mg/L_(broth). In some embodiments, cell viability is reduced by less than about any of 1.75-fold, 1.5-fold, 1.25-fold, 1-fold, 0.75-fold, 0.5-fold, or 0.25-fold. In some embodiments, cell viability is reduced by about 2-fold.

Cells in culture are also provided herein comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein the cells produce greater than about 400 nmole/g_(wcm)/hour of isoprene and carbon dioxide evolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In some embodiments, the isoprene produced is any concentration or amount disclosed in the section entitled “Exemplary Production of Isoprene.” In some embodiments, the amount of isoprene is between about any of 400 nmole/g_(wcm)/hour to 1 mole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 1 mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 40 mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 4 mmole/g_(wcm)/hour, 1 mmole/g_(wcm)/hour to 1.5 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour to 3 mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour to 5 mmole/g_(wcm)/hour, 5 mmole/g_(wcm)/hour to 25 mmole/g_(wcm)/hour, 25 mmole/g_(wcm)/hour to 100 mmole/g_(wcm)/hour, 100 mmole/g_(wcm)/hour to 500 mmole/g_(wcm)/hour, or 500 mmole/g_(wcm)/hour to 1000 mmole/g_(wcm)/hour. In some embodiments, the amount of isoprene is about any of 1 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour, 2 mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour, 4 mmole/g_(wcm)/hour, or 5 mmole/g_(wcm)/hour. In some embodiments, the carbon dioxide evolution rate is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

Provided herein are also cells in culture comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein cumulative total productivity of the isoprene produced by the cells in culture is greater than about 0.2 mg/L_(broth)/hour and carbon dioxide evolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In some embodiments, the cumulative total productivity of isoprene is any concentration or amount disclosed in the section entitled “Exemplary Production of Isoprene.” In some embodiments, the cumulative total productivity of the isoprene is between about any of 0.2 mg/L_(broth)/hour to 5 g/L_(broth)/hour, 0.2 mg/L_(broth)/hour to 1 g/L_(broth)/hour, 1 g/L_(broth)/hour to 2.5 g/L_(broth)/hour, 2.5 g/L_(broth)/hour to 5 g/L_(broth)/hour. In some embodiments, the carbon dioxide evolution rate is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

In addition, provided herein are cells in culture comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein peak concentration of the isoprene produced by the cells in culture is greater than about 10 ng/L_(broth) and carbon dioxide evolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In some embodiments, the peak concentration of isoprene is any concentration or amount disclosed in the section entitled “Exemplary Production of Isoprene.” In some embodiments, the peak concentration of isoprene is between about any of 10 ng/L_(broth) to 500 ng/L_(broth), 500 ng/L_(broth) to 1 μg/L_(broth), 1 μg/L_(broth) to 5 μg/L_(broth), 5 μg/L_(broth) to 50 μg/L_(broth), 5 μg/L_(broth) to 100 μg/L_(broth), 5 μg/L_(broth) to 250 μg/L_(broth), 250 μg/L_(broth) to 500 μg/L_(broth), 500 μg/L_(broth) to 1 mg/L_(broth), 1 mg/L_(broth) to 50 mg/L_(broth), 1 mg/L_(broth) to 100 mg/L_(broth), 1 mg/L_(broth) to 200 mg/L_(broth), 10 ng/L_(broth) to 200 mg/L_(broth), 5 μg/L_(broth) to 100 mg/L_(broth), or 5 μg/L_(broth) to 200 mg/L_(broth). In some embodiments, the peak concentration is any of about 10 ng/L_(broth), 100 ng/L_(broth), 1 μg/L_(broth), 5 μg/L_(broth), 1 mg/L_(broth), 30 mg/L_(broth), 100 mg/L_(broth), or 200 mg/L_(broth). In some embodiments, the carbon dioxide evolution rate is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

In some embodiments of any of the methods and cells described herein, carbon dioxide evolution rate and/or cell viability of a cell expressing a MVA pathway and/or DXP pathway RNA and/or protein from one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid is compared to a control cell lacking one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid. In some embodiments, carbon dioxide evolution rate and/or cell viability of a cell expressing a MVA pathway and/or DXP pathway RNA and/or protein from one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid under the control of an inducible promoter, wherein the promotor is induced, is compared to a control cell containing one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid under the control of an inducible promoter, wherein the promotor is not induced (uninduced). In some embodiments, the inducible promoter is a beta-galactosidase promoter.

In some embodiments, the methods of producing isoprene comprise: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein cells produce greater than about 400 nmole/g_(wcm)/hour of isoprene, and the carbon dioxide evolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour. Further provided herein are methods of producing isoprene comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the cumulative total productivity of the isoprene produced by the cells in culture is greater than about 0.2 mg/L_(broth)/hour and the carbon dioxide evolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour. Methods of producing isoprene are also provided herein comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the peak concentration of the isoprene produced by the cells in culture is greater than about 10 ng/L_(broth) and the carbon dioxide evolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In some embodiments of any of these methods, the carbon dioxide evolution rate is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about 50 mmol/L/hour or about 500 mmol/L/hour.

Further provided herein are cells in culture comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein the cells produce greater than about 400 nmole/g_(wcm)/hour of isoprene and carbon dioxide evolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour. Provided herein are also cells in culture comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein cumulative total productivity of the isoprene produced by the cells in culture is greater than about 0.2 mg/L_(broth)/hour and carbon dioxide evolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In addition, provided herein are cells in culture comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein peak concentration of the isoprene produced by the cells in culture is greater than about 10 ng/L_(broth) and carbon dioxide evolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In some embodiments of any of these cells in culture, the carbon dioxide evolution rate is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about 50 mmol/L/hour or about 500 mmol/L/hour.

Provided herein are also methods of producing isoprene comprising a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the liquid phase concentration of isoprene is less than about 200 mg/L and the cells produce greater than about 400 nmole/g_(wcm)/hour of isoprene. In some embodiments, the liquid phase concentration of isoprene in the culture is less than about any of 175 mg/L, 150 mg/L, 125 mg/L, 100 mg/L, 75 mg/L, 50 mg/L, 25 mg/L, 20 mg/L, 15 mg/L, 10 mg/L, 5 mg/L, or 2.5 mg/L. In some embodiments, the liquid phase concentration of isoprene in culture is between about any of 0.1 mg/L to 200 mg/L, 1 mg/L to 200 mg/L, 1 mg/L to 150 mg/L, 1 mg/L to 100 mg/L, 1 mg/L to 50 mg/L, 1 mg/L to 25 mg/L, 1 mg/L to 20 mg/L, or 10 mg/L to 20 mg/L.

Also provided herein are methods of producing a compound, wherein the compound has one or more characteristics selected from the group consisting of (a) a Henry's law coefficient of less than about 250 M/atm and (b) a solubility in water of less than about 100 g/L. In some embodiments, the method comprises: a) culturing cells under suitable conditions for production of the compound, wherein gas is added (such as the addition of gas to a system such as a fermentation system) at a gas sparging rate between about 0.01 vvm to about 2 vvm; and b) producing the compound. In some embodiments, the Henry's law coefficient of the compound is less than about any of 200 M/atm, 150 M/atm, 100 M/atm, 75 M/atm, 50 M/atm, 25 M/atm, 10 M/atm, 5 M/atm, or 1 M/atm. In some embodiments, the solubility in water of the compound is less than about any of 75 g/L, 50 g/L, 25 g/L, 10 g/L, 5 g/L, or 1 g/L. In some embodiments, the compound is selected from a group consisting of isoprene, an aldehyde (e.g., acetaldehyde), a ketone (e.g., acetone or 2-butanone), an alcohol (e.g., methanol, ethanol, 1-butanol, or C5 alcohols such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), an ester of an alcohol (e.g., ethyl acetate or acetyl esters of C5 alcohols), a hemiterpene, a monoterpene, a sesquiterpene, and C1 to C5 hydrocarbons (e.g., methane, ethane, ethylene, or propylene). In some embodiments, the C1 to C5 hydrocarbons are saturated, unsaturated, or branched. In particular embodiments, the compound is isoprene. In some embodiments of the methods of producing any of the compounds described above, the gas sparging rate is between about any of 0.1 vvm to 1 vvm, 0.2 vvm to 1 vvm, or 0.5 vvm to 1 vvm.

In one aspect, cells in culture are used to produce isoprene. In some embodiments, the cells in culture produce greater than about 400 nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/g_(wcm)/hr) of isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

In some embodiments, the cells in culture convert more than about 0.002% of the carbon in a cell culture medium into isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

In some embodiments, the cells in culture comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

In one aspect, described herein are methods of producing isoprene, such as methods of using any of the cells described herein to produce isoprene. In some embodiments, the method involves culturing cells under conditions sufficient to produce greater than about 400 nmole/g_(wcm)/hr of isoprene. In some embodiments, the method also includes recovering isoprene produced by the cells. In some embodiments, the method includes purifying isoprene produced by the cells. In some embodiments, the method includes polymerizing the isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions. In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD₆₀₀) during stationary phase is greater than or about 2 or more times the amount of isoprene produced during the growth phase for the same length of time. In some embodiments, the gas phase comprises greater than or about 9.5% (volume) oxygen, and the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit. In particular embodiments, (i) the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit, and (ii) the cells produce greater than about 400 nmole/g_(wcm)/hr of isoprene.

In some embodiments, the method includes culturing cells under conditions sufficient to convert more than about 0.002% of the carbon (mol/mol) in a cell culture medium into isoprene. In some embodiments, the method also includes recovering isoprene produced by the cells. In some embodiments, the method includes purifying isoprene produced by the cells. In some embodiments, the method includes polymerizing the isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

In some embodiments, isoprene is only produced in stationary phase. In some embodiments, isoprene is produced in both the growth phase and stationary phase. In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD₆₀₀) during stationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amount of isoprene produced during the growth phase for the same length of time.

In one aspect, described herein are compositions and systems that comprise isoprene. In some embodiments, the composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene. In some embodiments, the composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene (w/w) of the volatile organic fraction of the composition is isoprene.

In some embodiments, the composition comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition comprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition has less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared to the total weight of all C5 hydrocarbons in the composition. In particular embodiments, the composition has greater than about 2 mg of isoprene and has greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.

In some embodiments, the composition has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene. In particular embodiments, the composition also has greater than about 2 mg of isoprene.

In some embodiments, the composition has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms. In some embodiments, the composition has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of ethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more of the foregoing. In particular embodiments, the composition has greater than about 2 mg of isoprene and has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.

In some embodiments, the composition includes isoprene and one or more second compounds selected from the group consisting of 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. In various embodiments, the amount of one of these second components relative to the amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is at greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w).

In some embodiments, the composition comprises (i) a gas phase that comprises isoprene and (ii) cells in culture that produce greater than about 400 nmole/g_(wcm)/hr of isoprene. In some embodiments, the composition comprises a closed system, and the gas phase comprises greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg/L of isoprene when normalized to 1 mL of 1 OD₆₀₀ cultured for 1 hour. In some embodiments, the composition comprises an open system, and the gas phase comprises greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg/L of isoprene when sparged at a rate of 1 vvm. In some embodiments, the volatile organic fraction of the gas phase comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In some embodiments, the volatile organic fraction of the gas phase comprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In some embodiments, the volatile organic fraction of the gas phase has less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In particular embodiments, the volatile organic fraction of the gas phase has greater than about 2 mg of isoprene and has greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction.

In some embodiments, the volatile organic fraction of the gas phase has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a compound that inhibits the polymerization of isoprene for any compound in the volatile organic fraction of the gas phase that inhibits the polymerization of isoprene. In particular embodiments, the volatile organic fraction of the gas phase also has greater than about 2 mg of isoprene.

In some embodiments, the volatile organic fraction of the gas phase has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms. In some embodiments, the volatile organic fraction of the gas phase has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of ethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more of the foregoing. In particular embodiments, the volatile organic fraction of the gas phase has greater than about 2 mg of isoprene and has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.

In some embodiments, the volatile organic fraction of the gas phase has includes isoprene and one or more second compounds selected from the group consisting of 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. In various embodiments, the amount of one of these second components relative to amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is at greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w) in the volatile organic fraction of the gas phase.

In some embodiments of any of the compositions described herein, at least a portion of the isoprene is in a gas phase. In some embodiments, at least a portion of the isoprene is in a liquid phase (such as a condensate). In some embodiments, at least a portion of the isoprene is in a solid phase. In some embodiments, at least a portion of the isoprene is adsorbed to a solid support, such as a support that includes silica and/or activated carbon. In some embodiments, the composition includes ethanol. In some embodiments, the composition includes between about 75 to about 90% by weight of ethanol, such as between about 75 to about 80%, about 80 to about 85%, or about 85 to about 90% by weight of ethanol. In some embodiments, the composition includes between about 4 to about 15% by weight of isoprene, such as between about 4 to about 8%, about 8 to about 12%, or about 12 to about 15% by weight of isoprene.

In some embodiments, the systems include any of the cells and/or compositions described herein. In some embodiments, the system includes a reactor that chamber comprises cells in culture that produce greater than about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/g_(wcm)/hr isoprene. In some embodiments, the system is not a closed system. In some embodiments, at least a portion of the isoprene is removed from the system. In some embodiments, the system includes a gas phase comprising isoprene. In various embodiments, the gas phase comprises any of the compositions described herein.

In some embodiments of any of the compositions, systems, and methods described herein, a nonflammable concentration of isoprene in the gas phase is produced. In some embodiments, the gas phase comprises less than about 9.5% (volume) oxygen. In some embodiments, the gas phase comprises greater than or about 9.5% (volume) oxygen, and the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit. In some embodiments, the portion of the gas phase other than isoprene comprises between about 0% to about 100% (volume) oxygen, such as between about 10% to about 100% (volume) oxygen. In some embodiments, the portion of the gas phase other than isoprene comprises between about 0% to about 99% (volume) nitrogen. In some embodiments, the portion of the gas phase other than isoprene comprises between about 1% to about 50% (volume) CO₂.

In some embodiments of any of the aspects described herein, the cells in culture produce isoprene at greater than or about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/g_(wcm)/hr isoprene. In some embodiments of any of the aspects described herein, the cells in culture convert greater than or about 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6%, or more of the carbon in the cell culture medium into isoprene. In some embodiments of any of the aspects described herein, the cells in culture produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wet weight of the cells/hr (ng/g_(wcm)/h). In some embodiments of any of the aspects described herein, the cells in culture produce a cumulative titer (total amount) of isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/L of broth (mg/L_(broth), wherein the volume of broth includes the volume of the cells and the cell medium). Other exemplary rates of isoprene production and total amounts of isoprene production are disclosed herein.

In some embodiments of any of the aspects described herein, the cells further comprise a heterologous nucleic acid encoding an IDI polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding an IDI polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise a heterologous nucleic acid encoding a DXS polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding a DXS polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise one or more nucleic acids encoding an IDI polypeptide and a DXS polypeptide. In some embodiments of any of the aspects described herein, one nucleic acid encodes the isoprene synthase polypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments of any of the aspects described herein, one vector encodes the isoprene synthase polypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments, the vector comprises a selective marker, such as an antibiotic resistance nucleic acid.

In some embodiments of any of the aspects described herein, the heterologous isoprene synthase nucleic acid is operably linked to a T7 promoter, such as a T7 promoter contained in a medium or high copy plasmid. In some embodiments of any of the aspects described herein, the heterologous isoprene synthase nucleic acid is operably linked to a Trc promoter, such as a Trc promoter contained in a medium or high copy plasmid. In some embodiments of any of the aspects described herein, the heterologous isoprene synthase nucleic acid is operably linked to a Lac promoter, such as a Lac promoter contained in a low copy plasmid. In some embodiments of any of the aspects described herein, the heterologous isoprene synthase nucleic acid is operably linked to an endogenous promoter, such as an endogenous alkaline serine protease promoter. In some embodiments, the heterologous isoprene synthase nucleic acid integrates into a chromosome of the cells without a selective marker.

In some embodiments, one or more MVA pathway, IDI, DXP, or isoprene synthase nucleic acids are placed under the control of a promoter or factor that is more active in stationary phase than in the growth phase. For example, one or more MVA pathway, IDI, DXP, or isoprene synthase nucleic acids may be placed under control of a stationary phase sigma factor, such as RpoS. In some embodiments, one or more MVA pathway, IDI, DXP, or isoprene synthase nucleic acids are placed under control of a promoter inducible in stationary phase, such as a promoter inducible by a response regulator active in stationary phase.

In some embodiments of any of the aspects described herein, at least a portion of the cells maintain the heterologous isoprene synthase nucleic acid for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments of any of the aspects described herein, the nucleic acid comprising the isoprene synthase, IDI, or DXS nucleic acid also comprises a selective marker, such as an antibiotic resistance nucleic acid.

In some embodiments of any of the aspects described herein, the cells further comprise a heterologous nucleic acid encoding an MVA pathway polypeptide (such as an MVA pathway polypeptide from Saccharomyces cerevisia or Enterococcus faecalis). In some embodiments of any of the aspects described herein, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding an MVA pathway polypeptide (such as an MVA pathway polypeptide from Saccharomyces cerevisia or Enterococcus faecalis). In some embodiments of any of the aspects described herein, the cells comprise an isoprene synthase, DXS, and MVA pathway nucleic acid. In some embodiments of any of the aspects described herein, the cells comprise an isoprene synthase nucleic acid, a DXS nucleic acid, an IDI nucleic acid, and a MVA pathway nucleic (in addition to the IDI nucleic acid).

In some embodiments of any of the aspects described herein, the isoprene synthase polypeptide is a polypeptide from a plant such as Pueraria (e.g., Pueraria montana or Pueraria lobata) or Populus (e.g., Populus tremuloides, Populus alba, Populus nigra, Populus trichocarpa, or the hybrid, Populus alba x Populus tremula).

In some embodiments of any of the aspects described herein, the cells are bacterial cells, such as gram-positive bacterial cells (e.g., Bacillus cells such as Bacillus subtilis cells or Streptomyces cells such as Streptomyces lividans, Streptomyces coelicolor, or Streptomyces griseus cells). In some embodiments of any of the aspects described herein, the cells are gram-negative bacterial cells (e.g., Escherichia cells such as Escherichia coli cells, Rhodopseudomonas sp. such as Rhodopseudomonas palustris cells, Pseudomonas sp. such as Pseudomonas fluorescens cells or Pseudomonas putida cells, or Pantoea cells such as Pantoea citrea cells). In some embodiments of any of the aspects described herein, the cells are fungal, cells such as filamentous fungal cells (e.g., Trichoderma cells such as Trichoderma reesei cells or Aspergillus cells such as Aspergillus oryzae and Aspergillus niger) or yeast cells (e.g., Yarrowia cells such as Yarrowia lipolytica cells or Saccharomyces cells such as Saccharomyces cerevisiae).

In some embodiments of any of the aspects described herein, the microbial polypeptide carbon source includes one or more polypeptides from yeast or bacteria. In some embodiments of any of the aspects described herein, the plant polypeptide carbon source includes one or more polypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.

Production of Isoprene in Anaerobic Microorganisms Using Synthesis Gas as an Energy Source

In some embodiments, the bioisoprene composition is produced in anaerobic microorganisms using synthesis gas as an energy source as described in U.S. Provisional Patent Application Nos. 61/289,347 and 61/289,355, filed on Dec. 22, 2009, the disclosures of which are incorporated herein by reference in their entireties.

Production of isoprene from syngas by anaerobic organisms may provide a number of advantages over production of isoprene from sugars by aerobic organisms. First, the maximum theoretical mass yield of isoprene can be greater for the aerobic organisms, as discussed further below. Second, the anaerobic organisms do not have excess reducing power in the form of NAD(P)H that must be turned over via cell growth, formation of byproducts (such as glycerol, lactic acid, or ethanol) or oxidation using molecular oxygen. Without this NAD(P)H turnover requirement, anaerobic organisms can have higher energy yield, lower oxygen demand, lower heat of fermentation, and lower utility costs to run the process. Third, due to the lack of oxygen in the system, anaerobic organisms can have greater isoprene concentration in the offgas, lower probability of creating a flammable isoprene-oxygen mixture, easier recovery, and higher isoprene quality. Fourth, the anaerobic organisms can be more easily grown by using existing infrastructure, such as existing plants designed for production of bioethanol.

Anaerobic organisms useful for isoprene production can include obligate anaerobes, facultatitive anaerobes, and aerotolerant anaerobes. The obligate anaerobes can be any one or combination selected from the group consisting of Clostridium ljungdahlii, Clostridium autoethanogenum, Eurobacterium limosum, Clostridium carboxydivorans, Peptostreptococcus productus, and Butyribacterium methylotrophicum. Syngas useful as energy source for production of isoprene can be derived from a feedstock by a variety of processes, including methane reforming, coal liquefaction, co-firing, fermentative reactions, enzymatic reactions, and biomass gasification.

Exemplary Purification Methods

In some embodiments, any of the methods described herein further include recovering the co-produced compounds. In some embodiments, any of the methods described herein further include recovering the isoprene. In some embodiments, any of the methods described herein further include recovering the hydrogen by cryogenic membrane, adsorption matrix-based separation methods.

The isoprene and hydrogen produced using the compositions and methods described herein can be recovered using standard techniques. such as gas stripping, membrane enhanced separation, fractionation, adsorption/desorption, pervaporation, thermal or vacuum desorption of isoprene from a solid phase, or extraction of isoprene immobilized or absorbed to a solid phase with a solvent (see, for example, U.S. Pat. Nos. 4,703,007, 4,570,029, and 4,740,222 (“Recovery and Purification of Hydrogen from Refinery and Petrochemical Off-gas Streams”) which are each hereby incorporated by reference in their entireties, particularly with respect to isoprene recovery and purification methods ('007 and '029 patents) and with respect to hydrogen recovery and purification methods ('222 patent)). In particular embodiments, extractive distillation with an alcohol (such as ethanol, methanol, propanol, or a combination thereof) is used to recover the isoprene. In some embodiments, the recovery of isoprene involves the isolation of isoprene in a liquid form (such as a neat solution of isoprene or a solution of isoprene in a solvent). Gas stripping involves the removal of isoprene vapor from the fermentation off-gas stream in a continuous manner. Such removal can be achieved in several different ways including, but not limited to, adsorption to a solid phase, partition into a liquid phase, or direct condensation (such as condensation due to exposure to a condensation coil or do to an increase in pressure). In some embodiments, membrane enrichment of a dilute isoprene vapor stream above the dew point of the vapor resulting in the condensation of liquid isoprene. In some embodiments, the isoprene is compressed and condensed.

The recovery of isoprene may involve one step or multiple steps. In some embodiments, the removal of isoprene vapor from the fermentation off-gas and the conversion of isoprene to a liquid phase are performed simultaneously. For example, isoprene can be directly condensed from the off-gas stream to form a liquid. In some embodiments, the removal of isoprene vapor from the fermentation off-gas and the conversion of isoprene to a liquid phase are performed sequentially. For example, isoprene may be adsorbed to a solid phase and then extracted from the solid phase with a solvent.

The recovery of hydrogen may involve one step or multiple steps. In some embodiments, the removal of hydrogen gas from the fermentation off-gas and the conversion of hydrogen to a liquid phase are performed simultaneously. In some embodiments, the removal of hydrogen gas from the fermentation off-gas and the conversion of hydrogen to a liquid phase are performed sequentially. For example, hydrogen may be adsorbed to a solid phase and then desorbed from the solid phase by a pressure swing. In some embodiments, recovered hydrogen gas is concentrated and compressed.

In some embodiments, any of the methods described herein further include purifying the isoprene. For example, the isoprene produced using the compositions and methods described herein can be purified using standard techniques. Purification refers to a process through which isoprene is separated from one or more components that are present when the isoprene is produced. In some embodiments, the isoprene is obtained as a substantially pure liquid. Examples of purification methods include (i) distillation from a solution in a liquid extractant and (ii) chromatography. As used herein, “purified isoprene” means isoprene that has been separated from one or more components that are present when the isoprene is produced. In some embodiments, the isoprene is at least about 20%, by weight, free from other components that are present when the isoprene is produced. In various embodiments, the isoprene is at least or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%, by weight, pure. Purity can be assayed by any appropriate method, e.g., by column chromatography, HPLC analysis, or GC-MS analysis.

In some embodiments, any of the methods described herein further include purifying the hydrogen. For example, the hydrogen produced using the compositions and methods described herein can be purified using standard techniques. Purification refers to a process through which hydrogen is separated from one or more components that are present when the hydrogen is produced. In some embodiments, the hydrogen is obtained as a substantially pure gas. In some embodiments, the hydrogen is obtained as a substantially pure liquid. Examples of purification methods include (i) cryogenic condensation and (ii) solid matrix adsorption. As used herein, “purified hydrogen” means hydrogen that has been separated from one or more components that are present when the hydrogen is produced. In some embodiments, the hydrogen is at least about 20%, by weight, free from other components that are present when the hydrogen is produced. In various embodiments, the hydrogen is at least or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%, by weight, pure. Purity can be assayed by any appropriate method, e.g., by column chromatography or GC-MS analysis.

In some embodiments, at least a portion of the gas phase remaining after one or more recovery steps for the removal of isoprene is recycled by introducing the gas phase into a cell culture system (such as a fermentor) for the production of isoprene.

Methods and apparatus for the purification of a bioisoprene composition from fermentor off-gas is described in U.S. Provisional Patent Application No. 61/88,142, filed Dec. 18, 2009, which is incorporated herein by reference in its entirety.

A bioisoprene composition from a fermentor off-gas may contain bioisoprene with volatile impurities and bio-byproduct impurities. In some embodiments, a bioisoprene composition from a fermentor off-gas is purified using a method comprising: (a) contacting the fermentor off-gas with a solvent in a first column to form: an isoprene-rich solution comprising the solvent, a major portion of the isoprene and a major portion of the bio-byproduct impurity; and a vapor comprising a major portion of the volatile impurity; (b) transferring the isoprene-rich solution from the first column to a second column; and (c) stripping isoprene from the isoprene-rich solution in the second column to form: an isoprene-lean solution comprising a major portion of the bio-byproduct impurity; and a purified isopene composition.

FIG. 169 illustrates an exemplary method of purifying isoprene and an exemplary apparatus. Fermentor off-gas comprising isoprene may be generated from renewable resources (e.g., carbon sources) by any method in the art for example, as described in U.S. provisional patent application Nos. 61/187,944, the content of which is hereby incorporated by reference, particularly with respect to the methods of generating fermentor off-gas comprising isoprene. The fermentor off-gas generated from one or more individual fermentors 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more fermentors connected in series and/or in parallel) may be directed to a first column 14. As described below, the fermentor off-gas may be directed through an isolation unit 16 and/or compressed by a compression means, such as compression system 18. Additionally, the temperature of the fermentor off-gas may optionally be reduced at any point, for example, to form a condensate or partial condensate prior to contact with the solvent (which may aid in solubilization of one or more off-gas components, such as isoprene). The fermentor off-gas may be contacted (e.g., absorbed) at column 14 with a solvent (e.g., any solvent described herein, such as a non-polar high boiling-point solvent). The volatile impurities having less propensity for absorption in the solvent (particularly with non-polar high boiling-point solvents) are separated from the remaining solvent/fermentor off-gas mixture, resulting in a vapor comprising a major portion of the volatile impurity (e.g., exiting at port 20), and an isoprene-rich solution having a major portion of the isoprene and a major portion of the bio-byproduct impurity (e.g., at port 22). The solvent may optionally be heated by any suitable means (e.g., by steam) prior to, simultaneously, and/or after contact with the fermentor off-gas, which may aid in separation of the volatile impurity from the remaining solution. Steam may be directed through the column (at any suitable location, such as near entry of the off-gas and/or the opposite end of the volatile impurity exit as shown in FIG. 169) to provide a sweeping vapor phase which may aid in the removal of the volatile impurity.

The isoprene-rich solution having a major portion of the isoprene and a major portion of the bio-byproduct impurity (e.g., at port 22) may be directed to a second column 24. The second column may be isolated from the first column 14 (as shown in FIG. 169) or may be part of a single column comprising both the first and second columns (e.g., a tandem column wherein the solvent enters the first column at or near one end, and exits the second column at or near an opposite end). The isoprene may be stripped from the isoprene-rich solution in the second column to generate a purified isopene composition (e.g., at port 26) and an isoprene-lean solution comprising a major portion of the bio-byproduct impurity (e.g., at port 28). The isoprene-rich solution may be heated by any suitable means (e.g., by steam), which may aid in stripping of the isoprene from the remaining solution. Steam may be directed through the column (at any suitable location, such as the opposite end of the entry point of the isoprene-rich solution and/or the near the end of the isoprene-lean solution exit as shown in FIG. 169).

As described herein, the columns may be conventional and of any suitable size. Exemplary types of columns are commercially available from manufacturers including Koch Modular Process Systems (Paramus, N.J.), Fluor Corporation (Irving, Tex.), Kuhni USA (Mount Holly, N.C.). In general, columns are designed to maximize vapor/liquid contact in order to achieve the desired efficiency. This is achieved by filling the column with either a packing material, or trays spaced at regular intervals along the column. Suitable packing materials include both random and structured types based on metal, glass, polymer and ceramic materials. Exemplary random packing types include Raschig rings, Pall rings, A-PAK rings, Saddle rings, Pro-Pak, Heli-Pak, Ceramic saddles and FLEXIRINGS®. Structured packings include wire mesh and perforated metal plate type materials. Manufacturers specializing in column packings include ACS Separations & Mass-Transfer Products (Houston, Tex.), Johnson Bros. Metal Forming Co. (Berkeley, Ill.) and Koch Glitsch, Inc. Knight Div. (East Canton, Ohio). The efficiency of a gas stripping column is expressed in terms of the theoretical plate height and the total number of plates in the column. In general, the greater the number of theoretical plates present, the greater the efficiency of the column. Laboratory scale columns can be purchased from Ace Glass (Vineland, N.J.), Sigma-Aldrich (St. Louis, Mo.) and Chemglass (Vineland, N.J.). Suitable types of glass column include Vigreux, Snyder, Hemple and Perforated-plate type columns. Columns can include packing materials, or contain features designed to maximize vapor/liquid contact. A laboratory scale gas scrubber unit (part # CG-1830-10) is available from Chemglass and consists of a packed glass column, solvent reservoir and solvent recirculation pump.

The purified isoprene composition from the second column 24 (e.g., exiting at port 26) may be further purified by any suitable means (e.g., by using a reflux condenser 34 and/or an adsorption system 36, such as a silica adsorption system). The reflux reduces the solvent composition in the isoprene product. The isoprene-lean solution may be recycled back to the first column for reuse (e.g., as shown in FIG. 169 at port 30). The isoprene-lean solution may be purified by any suitable means (e.g., by liquid-liquid extraction and/or an adsorption system 32, such as a silica adsorption system) prior to recycling to the first column 14 to reduce to amount of bio-byproduct. Additionally, the temperature of the isoprene-lean solution may be reduced by any suitable means prior to recycling to the first column 14 (e.g., prior to, simultaneously, and/or after optionally purifying the isoprene solution). FIG. 169 shows an example of reducing the temperature of the isoprene-lean solution at port 40 prior to purification of the isoprene-lean solution (in this case, using coolant for temperature reduction).

The vapor comprising a major portion of the volatile impurity (e.g., the vapor exiting at port 20 in FIG. 169) may comprise a minor portion of isoprene (e.g., residual isoprene not remaining in the isoprene-rich solution). The residual isoprene may be recollected for use from the vapor comprising a major portion of the volatile impurity by any suitable means (e.g., an adsorption system 38, such as an activated carbon adsorption system) and in some cases, as shown in FIG. 169, may be combined with the purified isoprene composition (e.g., prior to, during, or after additional purification, such as an adsorption system similar to system 36). FIG. 169 also shows an optional capture device 42 (e.g., a thermal oxidizer and/or CO₂ capture system) capable of reducing the amount of undesirable components released into the atmosphere (e.g., CO₂) from the vapor.

Exemplary Chemical Transformations of Isoprene

Although current industrial use of isoprene is predominantly in the production of synthetic rubber, isoprene is a reactive conjugated diene and undergoes a varieties of chemical transformations to form oxygenates and higher molecular weight hydrocarbons. For example, Palladium(0) complexes (Pd(acac)₂-Ph₃P and Pd(OAc)₂-Ph₃P) catalyze dimerization and telomerization of isoprene in alcohol solvents to give linear isoprene dimers (e.g. 2,7-dimethyl-1,3,7-octatriene) and methoxydimethyloctadienes (Zakharkin, L. I. and Babich, S. A. Russ. Chem. Bull. (1976), pp 1967-1968.) Adams, J. M. and Clapp, T. V. (Clay and Clay Minerals (1986), 34(3), 287-294) reported reactions of isoprene over divalent and trivalent transition metal-exchanged montmorillonites (e.g. Cr³⁺-montmorillonite) to give isoprene dimers and adducts with methanol. The linear dimerization of isoprene catalyzed by Ni(0)-aminophosphinite systems resulted in regioselective tail-to-tail linear dimers, accompanied by a competitive cyclodimerization reaction (Denis, Philippe; Croizy, Jean Francois; Mortreux, Andre; Petit, Francis, Journal of Molecular Catalysis (1991), 68(2), 159-75. Denis, Philippe; Jean, Andre; Croizy, Jean Francois; Mortreux, Andre; Petit, Francis, Journal of the American Chemical Society (1990), 112(3), 1292-4.) New chiral aminophosphinite ligands, e.g., (+)-MeCH₂CHMeCH(NH₂)CH₂OPPh₂ was investigated as homogeneous catalysts in the linear dimerization of isoprene, leading to a conversion rate above 50% (Masotti, Henriette; Peiffer, Gilbert; Siv, Chhan; Courbis, Pierre; Sergent, Michelle; Phan Tan Luu, Roger, Bulletin des Societes Chimiques Belges (1991), 100(1), 63-77.)

Thermal dimerization of isoprene at 110-250° in presence of dinitrocresol as polymerization inhibitor gives high yields of dimers and little polymer (U.S. Pat. No. 4,973,787.) Ni-catalyzed dimerization of isoprene yields a dimethyl-1,5-cyclooctadiene mixture consisting of 80% 1,5-dimethyl-1,5-cyclooctadiene and 20% 1,6-dimethyl-1,5-cyclooctadiene (Doppelt, Pascal; Baum, Thomas H.; Ricard, Louis, Inorganic Chemistry (1996), 35(5), 1286-91.) Isoprene is converted to dimethylcyclooctadienes with a catalytic amt. of Cp*Ru(η4-isoprene)Cl and AgOTf (Itoh, Kenji; Masuda, Katsuyuki; Fukahori, Takahiko; Nakano, Katsumasa; Aoki, Katsuyuki; Nagashima, Hideo, Organometallics (1994), 13(3), 1020-9.) JP59065026A (1984) reported preparation of 1,6-dimethyl-1,5-cyclooctadiene by cyclic dimerization of isoprene in the presence of catalysts comprising Fe carboxylates or β-diketone compounds, organo-Al or Mg compounds, and 2,2′-dipyridyl derivatives having electron-donating groups. Dimethylcyclooctadiene was prepared by cyclodimerization of isoprene over 3-component catalysts containing Ni carboxylates or β-ketones, organoaluminum or organomagnesium compounds and substituted triphenylphosphite (JP58055434A, 1983.) 1,5-Dimethyl-1,5-cyclooctadiene was prepared by cyclodimerization of isoprene at 100-300° in an inert organic solvent in the presence of a homogeneous catalyst containing Fe(3) salt, organoaluminum compound and an activator (SU615056A1, 1978), in the presence of a homogeneous catalyst containing Ni acetylacetonate, a triarylphosphite and perhydroalumophenolene (SU493-455A1, 1975), in the presence of a catalyst containing a mixture of a Ni carboxylate or carboxylate or chelate compounds of Ni and 1-hydroxy-3-carbonyl compounds, trialkylaluminum, dialkylmagnesium or active organo-Mg compounds obtained from conjugated dienes and Mg, triaryl phosphites and tertiary amines (JP48064049A, 1973), or in the presence of a catalyst composed of Ni naphthenate, Et₃Al, and tri-o-cresyl phosphate (Suga, K.; Watanabe, S.; Fujita, T.; Shimada, T., Israel Journal of Chemistry (1972), 10(1), 15-18.) U.S. Pat. No. 3,954,665 disclosed dimerization of isoprene in the presence of reaction products of [(η3-C6H5)NiBr]₂ or [M(NO)₂X]₂ (M=Fe, Co; X═Cl, I, Br) with Fe, Co, or Ni carbonyls. European Patent No. 2411 (1981) disclosed cyclodimerization of isoprene over a Fe(NO)₂Cl-bis(1,5-cyclooctadiene)nickel catalyst at from −5° to +20° to give 1-methyl- and 2-methyl-4-isopropenyl-1-cyclohexene and 1,4- and 2,4-dimethyl-4-vinyl-1-cyclohexene. U.S. Pat. No. 4,189,403 disclosed preparation of 1,5-dimethyl-1,5-cyclooctadiene and 1,4-dimethyl-4-vinyl-1-cyclohexene by contacting isoprene with a mixed catalyst of a tris(substituted hydrocarbyl) phosphite, arsenite, or antimonite and a Group VIII metal(0) compound (e.g. Ni acetylacetonate). Jackstell, R.; Grotevendt, A.; Michalik, D.; El Firdoussi, L.; Beller, M. J. Organometallic Chem. (2007) 692(21), 4737-4744 cites the use of palladium/carbene catalysts for isoprene dimerization. Bowen, L.; Charernsuk, M.; Wass, D. F. Chem. Commun. (2007) 2835-2837 describes the use of a chromium N,N-bis(diarylphosphino)amine catalyst for the production of linear and cyclic trimers of isoprene.

Isoprene was reportedly dimerized in the presence of a Ni catalyst to yield cis-2-isopropenyl-1-methylvinylcyclobutane (Billups, W. E.; Cross, J. H.; Smith, C. V., Journal of the American Chemical Society (1973), 95(10), 3438-9.) The oligomerization of isoprene [78-79-5] catalyzed by nickel naphthenate and isoprenemagnesium in the presence of various phosphites as electron donors gave cyclic dimers containing dimethylcyclooctadiene [39881-79-3]; in particular 1,1,1-tris(hydroxymethyl)propane phosphite [39865-19-5] gave trimethylcyclododecatriene [39881-80-6] selectively (Suga, Kyoichi; Watanabe, Shoji; Fujita, Tsutomu; Shimada, Takashi, Journal of Applied Chemistry & Biotechnology (1973), 23(2), 131-8.) WO2006/051011 discloses preparation of trimethylcyclododecatriene, useful in perfumes and fragrances, by the trimerization of isoprene in the presence of a catalyst system comprising Ni and/or Ti, one or more organometallic compound, and a Group VA compound, and that the reaction is conducted in a hydroxyl group-containing solvent. Ligabue, R. A.; Dupont, J.; de Souza, R. F., Alegre, R. S. J. Mol. Cat. A: Chem. (2001), 169(1-2), 11-17, describes the selective dimerization of isoprene to six-membered dimers using an iron nitrosyl catalyst in an ionic liquid. Huchette, D.; Nicole, J.; Petit, F., Tetrahedron Letters (1979), (12), 1035-8, describes the electrochemical generation of an iron nitrosyl catalyst and subsequent use for the dimerization of isoprene to cyclohexene dimers. Zakharkin, L. I.; Zhigareva, G. G.; Pryanishnikov, A. P. Zhurnal Obshchei Khimii (1987), 57(11), 2551-6, describes the cyclooligomerization of isoprene on complex nickel and iron catalysts.

The highly pure isoprene starting compositions described herein are chemically transformed using each catalyst systems and reaction conditions disclosed in the references cited. Other catalysts and reaction conditions known in the art such as catalysts and reaction conditions applied to chemical transformations of 1,3-butadiene can be adapted to the isoprene starting compositions by one skilled in the art.

Dimerization and Trimerization

In some embodiments, a commercially beneficial amount of highly pure isoprene starting material undergoes catalytic chemical transformation to give dimers and trimers. Catalyst systems are identified using methods known in the art. Preferred catalysts include those known to convert isoprene to dimers and trimers with high efficiency. Examples include those based upon Palladium, Nickel, Cobalt, Iron and Chromium.

In some embodiments, a commercially beneficial amount of highly pure isoprene starting material undergoes thermal or catalytic dimerization. The isoprene starting composition is converted to a mixture of dimers that includes unsaturated 6 and 8-membered rings by heating the starting composition under pressure. In some embodiments, the starting isoprene composition is heated to over or about 100, 125, 150, 175, 200, 225 or 250° C. under pressure. In some embodiments, the starting isoprene composition is heated in the presence of an antioxidant (e.g. 2,6-di-tert-butyl-4-methylphenol) to prevent radical-mediated polymerization. In some embodiments, the reaction is accelerated by one or more catalysts selected from catalysts known in the art for catalyzing cyclic dimerization of isoprene, e.g., iron nitrosyl halide catalysts described in U.S. Pat. Nos. 4,144,278, 4,181,707, 5,545,789 and European patent EP0397266A2. The use of iron nitrosyl catalysts in ionic liquids is described by Ligabue et al. (2001) J. Mol. Catalysis. A: Chemical, 169 (2), 11-17. Examples of catalysts include but are not limited to Fe(NO)₂Cl₂ and Cr³⁺-montmorillonite. In another example, an isoprene starting composition is converted to C₁₀ cyclic dimers (e.g. a mixture of dimethyl-cyclooctadienes) using a ruthenium catalyst (Itoh, Kenji; Masuda, Katsuyuki; Fukahori, Takahiko; Nakano, Katsumasa; Aoki, Katsuyuki; Nagashima, Hideo, Organometallics (1994), 13(3), 1020-9.)

In a specific embodiment, a isoprene starting composition in a liquid state is heated to over 100° C. under pressure and in the presence of an antioxidant to produce a mixture of dimers that includes 6 and 8-membered rings, for examples, limonene (1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene), 1-methyl-5-(prop-1-en-2-yl)cyclohex-1-ene, 1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene, 1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta-1,5-diene, 2,6-dimethyl-1,3-cyclooctadiene, 2,6-dimethyl-1,4-cyclooctadiene, 3,7-dimethyl-1,5-cyclooctadiene, 3,7-dimethyl-1,3-cyclooctadiene, 3,6-dimethyl-1,3-cyclooctadiene and other 8-membered ring isoprene dimers as described in Organometallics (1994), 13(3), 1020-9. All stereoisomers of these compounds are contemplated. Conversion of isoprene to dimers is best performed in the absence of oxygen so as to avoid the production of undesirable reaction products.

In some embodiments, a commercially beneficial amount of highly pure isoprene starting material undergoes photo-dimerization to give 4-membered ring dimers. The isoprene starting composition is converted by light irradiation to a mixture comprising one or more of 1,2-di(prop-1-en-2-yl)cyclobutane, 1,3-di(prop-1-en-2-yl)cyclobutane, 1-methyl-1-vinyl-3-(prop-1-en-2-yl)cyclobutane, 1-methyl-1-vinyl-2-(prop-1-en-2-yl)cyclobutane, 1,3-dimethyl-1,3-divinylcyclobutane, 1,2-dimethyl-1,2-divinylcyclobutane and stereoisomers thereof. In some embodiments, the highly pure isoprene starting material is dimerized in the presence of a catalyst (e.g. a nickel catalyst) or a photosensitizer (e.g. benzophenone) to yield 4-membered ring dimers, e.g. cis-2-isopropenyl-1-methylvinylcyclobutane, in addition to 6- and 8-membered rings [See: Hammond, J. S.; Turro, N. J.; Liu, R. S. H. (1963) “Mechanisms of Photochemical Reactions in Solution. XVI. Photosensitized Dimerization of Conjugated Dienes. J. Org. Chem., 28, 3297-3303.]

In some embodiments, a commercially beneficial amount of highly pure isoprene starting material is converted to a cyclic trimer in the presence of a catalyst system. In some embodiments, the cyclic trimer is trimethylcyclododecatriene. In some embodiments, the catalyst system comprises a nickel catalyst. In some embodiments, the catalyst system comprises a titanium compound. In some embodiments, the catalyst system comprises a nickel catalyst and a titanium compound. In some embodiments, the catalyst system comprises one or more organometallic compound and a Group VA compound. In some embodiments, the catalytic transformation is conducted in a hydroxyl group-containing solvent. In a particular embodiment, a starting isoprene composition is converted to trimethylcyclododecatriene in the presence of a catalyst system comprising Ni and/or Ti, one or more organometallic compound, and a Group VA compound, and that the reaction is conducted in a hydroxyl group-containing solvent.

In some embodiments, a commercially beneficial amount of highly pure isoprene starting material undergoes thermal or catalytic conversion to hydrocarbons in the C7 to C14 range by treatment with a C2 to C5 unsaturated hydrocarbon. The C2 to C5 hydrocarbon can be an alkene, a diene or an alkyne. For example, in some embodiments, isoprene is contacted with ethylene and an appropriate catalyst to produce C7 compounds. In another embodiment, isoprene is contacted with 1,3-butadiene to form cyclic C9 hydrocarbons. Hydrogenation of these C7 to C14 hydrocarbons results in compositions suitable for use as jet fuels and other aviation fuels. In yet another embodiment, unsaturated C7 to C14 hydrocarbons derived from isoprene and one or more unsaturated hydrocarbons undergo dehydrogenation to form aromatic derivatives suitable for use as jet fuels and other aviation fuels, or as blendstocks for such fuels.

In some embodiments, a commercially beneficial amount of highly pure isoprene starting material is converted to a mixture of linear dimers and trimers using a catalyst system in alcohol solvents. This reaction also produces alkoxy derivatives when performed in methanol or ethanol. In some embodiments, the catalyst system is a palladium-based catalyst system, e.g., a palladium acetylacetonate-triphenylphosphine system or a palladium acetate-triphenylphosphine system. In some embodiments, the alcohol solvent is methanol, ethanol or isopropyl alcohol. The nature of the products depends on the catalyst and the solvent used. For example, when a palladium-based catalyst system, e.g., Pd(acac)₂-Ph₃P or Pd(OAc)₂-Ph₃P, is used in isopropyl alcohol solvent, linear dimers of isoprene is formed, e.g. 2,7-dimethyl-1,3,7-octatriene and 2,7-dimethyl-2,4,6-octatriene. When this reaction is performed in methanol, methoxydimethyloctadienes (e.g., 1-methoxy-2,7-dimethyl-2,7-octadiene and 3-methoxy-2,7-dimethyl-1,7-octadiene) are formed in addition to linear isoprene dimers such as dimethyloctatrienes. Some reactions produce linear trimers of isoprene, such as a-farnesene (3,7,11-trimethyl-1,3,6,10-dodecatetraene), β-farnesene (7,11-dimethyl-3-methylene-1,6,10-dodecatriene) and other positional isomers (e.g. from tail to tail and head to head addition of isoprene units). In another example, isoprene is converted to linear and cyclic C15 trimers using a chromium N,N-bis(diarylphosphino)amine catalyst (Bowen, L.; Charernsuk, M.; Wass, D. F. Chem. Commun. (2007) 2835-2837.)

Conversion to Oxygenates

In some embodiments, a commercially beneficial amount of highly pure isoprene starting material is converted to fuel oxygenates by reaction with ethanol and other alcohols in the presence of an acid catalyst. In one embodiment, the acid catalyst is sulfuric acid. In another embodiment, the acid catalyst is a solid phase sulfuric acid (e.g., Dowex Marathon®). Other catalysts include both liquid and solid-phase fluorosulfonic acids, for example, trifluoromethanesulfonic acid and Nafion-H (DuPont). Zeolite catalysts can also been used, for example beta-zeolite, under conditions similar to those described by Hensel et al. [Hensen, K.; Mahaim, C.; Holderich, W. F., Applied Catalysis A: General (1997) 140(2), 311-329.] for the methoxylation of limonene and related monoterpenes. In some embodiments, a highly pure isoprene starting material is converted to alcohols and esters by a hydroxylation esterification process or other known reaction of alkenes in the art, for example peroxidation to epoxides with peracids such as peracetic acid and 3-chloroperbenzoic acid; and hydration to give alcohols and diols with i) water and acid catalysts and ii) hydroboration methods. Such reactions are described in, for example, Michael B. Smith and Jerry March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Sixth Edition, John Wiley & Sons, 2007. FIGS. 3 and 4 show examples of alcohols and oxygenates that can be produced from the starting isoprene compositions.

Partial Hydrogenation

In some embodiments, a commercially beneficial amount of highly pure isoprene starting material is partially hydrogenated to a mono-olefin (e.g. 2-methylbut-1-ene, 3-methyl-but-1-ene and 2-methylbut-2-ene). In some embodiments, the mono-olefin undergoes dimerization or reaction with other olefins using traditional hydrocarbon cationic catalysis, such as that used to covert isobutylene to isooctane. See, for example, H. M. Lybarger. Isoprene in Kirk-Othmer Encyclopedia of Chemical Technology, 4th ed., Wiley, New York (1995), 14, 934-952. In some preferred embodiments, the highly pure isoprene starting material is a bioisoprene composition.

In some embodiments, a commercially beneficial amount of highly pure isoprene starting material undergoes partial hydrogenation to form mono-olefins (e.g. 2-methylbut-1-ene, 3-methyl-but-1-ene and 2-methylbut-2-ene). In some preferred embodiments, the highly pure isoprene starting material is a bioisoprene composition. In some embodiments, partial hydrogenation of the isoprene starting material produces high yields of mono-olefins with minimal conversion to isopentane and low residual isoprene levels. In some embodiments, a mixture of mono-olefin and isopentane is produced, with low levels of residual isoprene. In some embodiments, the partial hydrogenation is selective hydrogenation where a particular mono-olefin such as 2-methylbut-2-ene, 2-methylbut-1-ene, or 3-methylbut-1-ene is preferentially produced. Preferred hydrogenation catalysts give high catalytic activity, maintain catalytic activity over time, and are highly selective for the conversion of isoprene to mono-olefins.

Suitable catalysts for partial hydrogenation of the isoprene starting composition may contain a platinum-group metal such as platinum, palladium, rhodium, or ruthenium, or a transition metal such as nickel, cobalt, copper, iron, molybdenum, or a noble metal such as silver or gold in elemental form, or a salt or complex with organic and inorganic ligands. Alloys of these metals can also be used in some circumstances. These metals and their derivatives can be pure or mixed with other active and inert materials. Catalysts may be adsorbed to a support material (such as activated carbon, alumina, or silica) in order to maximize the effective surface area. The physical morphology of hydrogenation catalysts is known to have a considerable influence on their performance. Preferred hydrogenation catalysts include heterogeneous palladium catalysts such as palladium on carbon (Pd/C), palladium on alumina (Pd/Al₂O₃), or palladium on silica (Pd/SiO₂) in grades ranging from 0.1% Pd to 20% Pd (w/w) relative to the support material. An example of a suitable selective hydrogenation catalyst is LD 2773, a sulfur tolerant promoted Pd on alumina (Axens, Rueil-Malmaison Cedex, France). Partial hydrogenation catalysts that allow the conversion of isoprene into isoamylenes with minimal conversion to isopentane include the Lindlar catalyst (Pd/BaSO₄ treated with quinoline), Pd/C treated with the triphenyl derivative of a group 15 element (N, P, As), Pd/C treated with a sulfur-containing compound, molybdenum sulfide, and Pd/Fe alloys. One preferred catalyst comprises palladium adsorbed to egg-shell alumina (d-Al₂O₃). Catalysts used in the refining industry for the removal of diolefins and alkynes from pyrolysis gasoline are particularly preferred, for example Ni/Al₂O₃ and Pd/Al₂O₃ based catalysts. Another suitable class of catalyst for the conversion of isoprene to isoamylenes are those used in industry for the hydrotreating of pyrolysis gasoline. See for example U.S. Pat. Nos. 7,014,750 and 6,9949,686, and references cited therein. In general, the catalysts used in pyrolysis gas hydrotreament allow for selective conversion of acetylyenes and diolefins (e.g. isoprene and piperylenes) into monoolefins.

The hydrogen source can be hydrogen gas or a hydrogen source including, but not limited to, hydrogen gas (H₂), formic acid, hydrazine, or isopropanol. The hydrogen source can be either chemically or biologically derived. In some embodiments the hydrogen used for hydrogenation is co-produced with isoprene during fermentation. The hydrogenation can be performed at hydrogen pressures ranging from 0.5 atm to 200 atm, or higher. The temperature can range from 0° C. to 200° C.

Products from partial hydrogenation of a bioisoprene composition are expected to contain certain impurities originally present in the starting isoprene composition and/or hydrogenated derivatives of the impurities such as acetone, ethanol, amyl alcohol, ethyl acetate, isoamyl acetate, methyl ethyl ketone and other saturated polar impurities.

In some embodiments, the isoprene starting composition undergoes partial hydrogenation or selective hydrogenation in the presence of a palladium catalyst. For example, palladium catalysts, such as Pd/CaCO₃, Pd/BaSO₄, Pd/C, Pd black, Pd/SiO₃, Pd/Al₂O₃, or Pd/SiO₂, were shown to convert isoprene to mono-olefins with a selectivity of greater than 95% over fully reduced C5 alkanes. Use of these palladium catalysts gave a mixture of mono-olefin products, with 5% Pd/CaCO₃ having the greatest selectivity for 3-methylbut-1-ene and 5% Pd/SiO₂ having the greatest selectivity for 2-methylbut-2-ene. (See G. C. Bond and A. F. Rawle. J. Mol. Catalysis. A: Chemical 109 (1996) 261-271.) Bond and Rawle have also shown that palladium-gold and palladium-silver catalysts (e.g., Pd—Au/SiO₂ and Pd—Ag/SiO₂) have high selectivity for reducing isoprene to mono-olefins over fully reduced C5 alkanes. In some embodiments, silica-supported polyamidoamine (PAMAM) dendrimer-palladium complexes have been used to selectively catalyze the reduction of cyclic and acyclic dienes to a mixture of mono-olefin isomers. Selectivity for the various mono-olefin isomers was dependent on the precise PAMAM ligand used in the catalyst. (See P. P. Zweni and H. Alper. Adv. Synth. Catal. 348 (2006) 725-731.) In some embodiments, reduction of isoprene to mono-olefins can be carried out using a Group VIB metal on an inorganic support (e.g., a metal zeolite) as a catalyst. For example, a Mo/Al₂O₃ catalyst was used to selectively reduce 1,3-butadiene to the respective mono-olefins. (See U.S. Pat. No. 6,235,954 B1.) In some embodiment, isoprene can be reduced to mono-olefins using a Group VIII metal catalyst promoted by a metal from Group IB, VIIB, VIIB, or zinc to reduce poisoning of the catalyst. (See U.S. Pat. No. 6,949,686 B2). In some embodiments, isoprene can be reduced to mono-olefins using a monolithic catalyst bed, which may be in a honeycomb configuration. Catalyst support materials for the monolithic catalyst bed may include metals such as nickel, platinum, palladium, rhodium, ruthenium, silver, iron, copper, cobalt, chromium, iridium, tin, and alloys or mixtures thereof. (See U.S. Pat. No. 7,014,750 B2). In some embodiments, isoprene can be reduced to mono-olefins using an eggshell Pd/d-Al₂O₃ catalyst, particularly if the reaction is free of water. The eggshell Pd/d-Al₂O₃ catalyst is selective for mono-olefins over fully reduced alkanes and 2-methylbut-2-ene is the thermodynamically favorable isomer. (See J.-R. Chang and C.-H. Cheng. Ind. Eng. Chem. Res. 36 (1997) 4094-4099.)

Light olefins (C3-C6) can be readily dimerized using acid catalysts to give higher olefins (C6-C12) also known as dimate. For example, the conversion of isobutylene (2-methylpropene) to isooctene is well described in the art using a variety of acid catalysts including sulfuric, phosphoric and other mineral acids, sulfonic acids, fluorosulfonic acids, zeolites and acidic clays.

In some embodiments, the isoprene starting composition undergoes partial hydrogenation or selective hydrogenation to form a mono-olefin, and the mono-olefin undergoes dimerization or reaction with other olefins using traditional hydrocarbon cationic catalysis, such as that used to covert isobutylene to isooctane, e.g. sulfuric, phosphoric and other mineral acids, sulfonic acids, fluorosulfonic acids, zeolites and acidic clays. See, for example, H. M. Lybarger. Isoprene in Kirk-Othmer Encyclopedia of Chemical Technology, 4th ed., Wiley, New York (1995), 14, 934-952. In some preferred embodiments, the highly pure isoprene starting material is a bioisoprene composition. Acid catalyst in both liquid and solid forms can be used. Acid resins are preferred and include Amberlyst 15, 35, XE586, XN1010 (Rohm and Haas) and similar acidic ion-exchange resins. Acidic molecular sieves are also preferred catalysts including a medium-pore acid molecular sieve such as ZSM-5, ferrierite, ZSM-22 and ZSM-23.

In some embodiments, the isoprene starting composition undergoes partial hydrogenation or selective hydrogenation to form isoamylenes. In some embodiments, the isoamylenes are dimerized to give C10 dimates such as isodecenes. In some embodiments, the isoamylenes are dimerized with an olefin such as propylene, butane or isobutene to give C8-C10 dimates. In some embodiments, the mono-olefin undergoes dimerization using a resin catalyst (e.g., Amberlyst, Amberlyst 35, Amberlyst 15, Amberlyst XN1010, Amberlyst XE586) to produce diisoamylenes. Use of such resin catalysts has been shown to minimize cracking and further oligomerization reactions, and under optimal conditions the resin catalysts provide selectivity for dimers of greater than 92%, with trimer formation of less than 8%. See, for example, M. Marchionna et al. Catalysis Today 65 (2001) 397-403. In some embodiments, the mono-olefin undergoes dimerization using a catalytic material containing an acidic mesoporous molecular sieve, such as a mesoporous sieve embedded in a zeolite structure, ZSM-5, ferrierite, ZSM-22, or ZSM-23. In a particular embodiment, the catalytic material is thermally stable at high temperatures (e.g., at least 900° C.). In another particular embodiment, the mono-olefin is substantially free of multi-unsaturated hydrocarbons, such as isoprene. Use of a catalytic material containing an acidic mesoporous molecular sieve for dimerization can lead to selectivity for dimers in excess of 80%. See, for example, US 2007/0191662 A1. In some embodiments, the mono-olefin undergoes dimerization using a solid acidic catalyst (e.g., a solid phosphoric acid catalyst, acidic ion exchange resins). Selectivity for dimers using a solid phosphoric acid catalyst can be at least 75%, at least 85% or at least 90%. See, for example, US 2009/0099400 A1 and U.S. Pat. No. 6,660,898 B1.

Efficient dimerization can sometimes require the presence of a polar component(s) such as water and oxygenated compound in the feed stream. Examples include alcohols such as methanol, ethanol and t-butanol, ethers such as methyl t-butyl ether (MTBE) and methyl t-amyl ether (TAME) or an ester such as C1 to C5 acetates. In some embodiments, isoamylenes can be converted to ethers such as TAME by treatment with alcohols in the presence of an acid catalyst.

In some embodiments, the C10 dimers of mono-olefins produced by any of the methods described herein can be reduced to fully saturated C10 alkylates (e.g., by hydrogenation). In a particular embodiment, isoprene or a mono-olefin can be reduced to an alkylate using a catalyst containing an acid component, such as sulfuric acid, a fluorosulfonic acid, a perhaloalkylsulfonic acid, an ionic liquid, or a mixture of Bronsted acids and Lewis acids, mixed with a polymer component, such as a polyacrylate. (See US 2010/0094072 A1).

In some embodiments, a commercially beneficial amount of highly pure isoprene starting material undergoes oligomerization in the presence of an acid catalysts to produce dimers, trimers, higher oligomers, aromatic products, and/or polymeric products. Kinetic control of the reaction can favor certain products, for example lower oligomers, although gum formation and coking are known issues leading to catalyst deactivation.

Exemplary Unsaturated Isoprene Derivatives

In some embodiments, the compositions and systems for producing a fuel constituent from isoprene further comprise an unsaturated hydrocarbon or oxygenate intermediate for chemical transformation of an isoprene starting composition to a fuel constituent. In some embodiment, the unsaturated hydrocarbon intermediate comprise one or more unsaturated dimers of isoprene selected from the group consisting of 1,2-di(prop-1-en-2-yl)cyclobutane, 1,3-di(prop-1-en-2-yl)cyclobutane, 1-methyl-1-vinyl-3-(prop-1-en-2-yl)cyclobutane, 1-methyl-1-vinyl-2-(prop-1-en-2-yl)cyclobutane, 1,3-dimethyl-1,3-divinylcyclobutane, 1,2-dimethyl-1,2-divinylcyclobutane, 1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene), 1-methyl-5-(prop-1-en-2-yl)cyclohex-1-ene, 1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene, 1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta-1,5-diene, 1,4-dimethyl-4-vinyl-1-cyclohexene, 2,4-dimethyl-4-vinyl-1-cyclohexene, 2,7-dimethyl-1,3,7-octatriene, 2,7-dimethyl-2,4,6-octatriene, 2,6-dimethyl-1,3-cyclooctadiene, 2,6-dimethyl-1,4-cyclooctadiene, 3,7-dimethyl-1,5-cyclooctadiene, 3,7-dimethyl-1,3-cyclooctadiene and 3,6-dimethyl-1,3-cyclooctadiene. In some embodiments, the unsaturated hydrocarbon intermediate comprises one or more unsaturated trimers such as a-farnesene, β-farnesene, trimethylcyclododecatrienes (e.g. 1,5,9-trimethyl-(1E,5E,9E)-cyclododecatriene and positional and geometric isomers thereof) and trimethyldodecatetraenes and the like. In some embodiments, the unsaturated oxygenate intermediate comprise one or more unsaturated methyl ethers such as 1-methoxy-2,7-dimethyl-2,7-octadiene and 3-methoxy-2,7-dimethyl-1,7-octadiene and the like. In some embodiments, the unsaturated oxygenate intermediate comprise one or more unsaturated ethyl ethers such as ethyl 3-methyl-3-butenyl ether, ethyl 1,1-dimethyl-2-propenyl ether, ethyl 1,2-dimethyl-2-propenyl ether, ethyl 2-methyl-3-butenyl ether and the like. In some embodiments, the unsaturated oxygenate intermediate comprise one or more unsaturated alcohols such as 3-methyl-3-buten-1-ol, 2-methyl-3-buten-2-ol, 3-methyl-3-buten-2-ol, 2-methyl-3-buten-1-ol and the like. In some embodiments, the unsaturated oxygenate intermediate comprise one or more unsaturated esters such as 3-methyl-3-buten-1-yl acetate, 2-methyl-3-buten-2-yl acetate, 3-methyl-3-buten-2-yl acetate, 2-methyl-3-buten-1-yl acetate and esters of other C₃-C₁₈ aliphatic carboxylic acids. In some embodiment, the unsaturated hydrocarbon intermediate comprise one or more isoamylenes, e.g. 2-methylbut-1-ene, 3-methyl-but-1-ene and 2-methylbut-2-ene. In some embodiments, the unsaturated hydrocarbon intermediate comprise on or more diisoamylenes derived from dimerization of isoamylenes.

Exemplary Hydrogenation of Unsaturated Intermediates

The unsaturated isoprene derivatives are subject hydrogenation in the presence of a hydrogenation catalyst to produce saturated compounds. The saturated compounds are characterized and their values as fuels are assessed. In some embodiments, the hydrogen source for hydrogenation is hydrogen gas. In some embodiments, the hydrogen gas is co-produced with the bioisoprene as described in U.S. provisional patent application No. 61/141,652, filed on Dec. 30, 2008 and US 2009/0203102 A1. In some embodiments, the hydrogenation catalyst is a palladium based catalyst such as Pd/C (e.g. 5% (wt.) Pd/C). In some embodiments, the hydrogenation catalyst is a Raney Nickel catalyst. In some embodiments, the hydrogenation catalyst is a homogenous catalyst such as ruthenium or rhodium based homogenous hydrogenation catalysts. In some embodiments, unsaturated isoprene dimers and trimers are hydrogenated to produce saturated C10 and C15 hydrocarbons suitable for making fuels. In some embodiments, unsaturated cyclic dimers are hydrogenated to produce saturated cyclic C10 hydrocarbons such as 1,2-bis(isopropyl)cyclobutane, 1,2-bis(isopropyl)cyclobutane, 1-methyl-4-isopropylcyclohexane, 1-methyl-3-isopropylcyclohexane, 1-ethyl-1,4-dimethylcyclohexane, 1-ethyl-1,3-dimethylcyclohexane, 1,5-dimethylcyclooctane and 1,4-dimethylcyclooctane. In some embodiments, unsaturated cyclic trimers are hydrogenated to produce saturated cyclic C15 hydrocarbons such as 1,5,9-trimethylcyclododecane and 1,5,10-trimethylcyclododecane (see Scheme VII). In some embodiments, unsaturated linear dimers are hydrogenated to produce saturated aliphatic C10 hydrocarbons such as 2,6-dimethyloctane, 2,7-dimethyloctane and 3,6-dimethyloctane. In some embodiments, unsaturated linear trimers are hydrogenated to produce saturated aliphatic C15 hydrocarbons such as 2,6,10-trimethyldodecane, 2,7,10-trimethyldodecane and 3,7,10-trimethyldodecane (see Scheme VIII). In some embodiments, the product of the dimerization of isoamylenes (diamylenes or C10 dimates) are fully hydrogenated to isoparaffins (e.g. 2,3,4,4-tetramethylhexane, 2,2,3,4-tetramethylhexane, 2,3,3,4-tetramethylhexane and 3,3,5-trimethylheptane) (see Scheme VIIIa). In some embodiments, a commercially beneficial amount of highly pure isoprene starting composition is hydrogenated to produce a product comprising 2-methylbutane. In some embodiments, the unsaturated isoprene hydroxylates are hydrogenated to produce saturated hydroxylates such as C5 alcohols and diols (e.g. 3-methyl-butan-1-ol, 2-methyl-butan-1-ol and 2-methyl-butan-2-ol, 3-methyl-butan-1,3-diol and 2-methyl-butan-2,3-diol), C-10 alcohols and diols (e.g. 3,7-dimethyloctan-1-ol, 2,7-dimethyloctan-1-ol, 2,7-dimethyloctan-2-ol and 2,7-dimethyloctan-2,7-diol) and cyclic C-10 alcohols (e.g. 2-(4-methylcyclohexyl)propan-2-ol, 2-(4-methylcyclohexyl)propan-1-ol, 2-(1,4-dimethylcyclohexyl)ethanol and 4-ethyl-1,4-dimethylcyclohexanol) (see Scheme IX). In some embodiments, the unsaturated isoprene oxygenates are hydrogenated to produce saturated ethers such as 1,3-diethoxy-3-methylbutane, 1-ethoxy-3-methylbutane, 1-methoxy-2,7-dimethyloctane and 3-methoxy-2,7-dimethyloctane (see Scheme X). It is understood that when an alkene moiety is hydrogenated, one or more stereo isomers are produced. The relative ratios between the stereo isomers depend on the reaction conditions and the catalysts used. When applicable, each and every stereo isomer is intended for the saturated hydrocarbons and oxygenates described herein.

In some embodiment, the fuel constituent produced by chemical transformation of a commercially beneficial amount of highly pure isoprene starting composition comprises saturated isoprene derivatives. In some embodiments, the fuel constituent comprises saturated C10 and C15 hydrocarbons derived from isoprene. In some embodiments, the fuel constituent comprises one or more saturated cyclic C10 hydrocarbons selected from the group consisting of 1,2-bis(isopropyl)cyclobutane, 1,2-bis(isopropyl)cyclobutane, 1-methyl-4-isopropylcyclohexane, 1-methyl-3-isopropylcyclohexane, 1-ethyl-1,4-dimethylcyclohexane, 1-ethyl-1,3-dimethylcyclohexane, 1,5-dimethylcyclooctane and 1,4-dimethylcyclooctane. In some embodiments, the fuel constituent comprises one or more saturated cyclic C15 hydrocarbons selected from the group consisting of 1,5,9-trimethylcyclododecane and 1,5,10-trimethylcyclododecane. In some embodiments, the fuel constituent comprises one or more saturated aliphatic C10 hydrocarbons selected from the group consisting of 2,6-dimethyloctane, 2,7-dimethyloctane and 3,6-dimethyloctane. In some embodiments, the fuel constituent comprises one or more saturated aliphatic C15 hydrocarbons selected from the group consisting of 2,6,10-trimethyldodecane, 2,7,10-trimethyldodecane and 3,7,10-trimethyldodecane. In some embodiments, the fuel constituent comprises 2-methylbutane. In some embodiments, the fuel constituent comprises one or more isoparaffins selected from the group consisting of 2,3,4,4-tetramethylhexane, 2,2,3,4-tetramethylhexane, 2,3,3,4-tetramethylhexane and 3,3,5-trimethylheptane. In some embodiments, the fuel constituent comprises one or more saturated hydroxylates selected from the group consisting of 3-methyl-butan-1-ol, 2-methyl-butan-1-ol and 2-methyl-butan-2-ol, 3-methyl-butan-1,3-diol, 2-methyl-butan-2,3-diol, 3,7-dimethyloctan-1-ol, 2,7-dimethyloctan-1-ol, 2,7-dimethyloctan-2-ol, 2,7-dimethyloctan-2,7-diol, 2-(4-methylcyclohexyl)propan-2-ol, 2-(4-methylcyclohexyl)propan-1-ol, 2-(1,4-dimethylcyclohexyl)ethanol and 4-ethyl-1,4-dimethylcyclohexanol. In some embodiments, the fuel constituent comprises one or more saturated ethers selected from the group consisting of 1,3-diethoxy-3-methylbutane, 1-ethoxy-3-methylbutane, 1-methoxy-2,7-dimethyloctane and 3-methoxy-2,7-dimethyloctane. In some embodiments, the fuel constituent comprises one or more oxygenates of isoprene selected from the group consisting of 3-methyltetrahydrofuran, 3-methyl-2-butanone and isopentyl acetate.

In some embodiments, the compositions and systems for producing a fuel constituent from isoprene further comprise catalysts for catalyzing the chemical transformation of an isoprene starting composition to a fuel constituent or an intermediate for making a fuel constituent. In some embodiments, the compositions and systems for producing a fuel constituent from isoprene further comprise catalysts for catalyzing hydrogenation of an unsaturated intermediate to produce a saturated fuel constituent. In some embodiments, the catalyst is any catalyst described or a combination of one or more of the catalyst described herein.

Method and/or Processes for Producing Fuels

The invention provides methods and/or processes for producing a fuel constituent from isoprene comprising: (a) obtaining a commercially beneficial amount of highly pure isoprene; and (b) chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent. In one embodiment, a highly pure isoprene composition is transformed to a fuel component in a continuous chemical process. In another embodiment, a highly pure isoprene is further purified before chemical transformation to a fuel composition. In yet another embodiment, a highly pure isoprene is chemically transformed to an intermediate composition; the intermediate composition undergoes further chemical transformations to produce a fuel or a fuel component. In a further embodiment, the fuel component produced is mixed with a petroleum distillate and other optional additives to make a fuel. In some preferred embodiments, the highly pure isoprene is a bioisoprene composition.

In some embodiments, the method for producing a fuel constituent from isoprene comprises obtaining a commercially beneficial amount of highly pure isoprene composition. In some embodiments, the highly pure isoprene composition useful in the invention comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene. In some embodiments, the highly pure isoprene composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene. In some embodiments, the starting isoprene composition comprises greater than or about 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 kg of isoprene.

In some embodiments, the method for producing a fuel constituent from isoprene comprises obtaining a commercially beneficial amount of highly pure isoprene composition. In some embodiments, the highly pure isoprene starting composition comprises greater than or about 98.0, 98.5, 99.0, 99.5, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the starting composition. In some embodiments, the highly pure isoprene composition useful in the invention comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the highly pure isoprene composition comprises less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the highly pure isoprene composition has less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared to the total weight of all C5 hydrocarbons in the composition. In particular embodiments, the highly pure isoprene composition has greater than about 2 mg of isoprene and has greater than or about 98.0, 98.5, 99.0, 99.5, 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.

In some embodiments, the method for producing a fuel constituent from isoprene comprises obtaining a commercially beneficial amount of highly pure isoprene composition. In some embodiments, the highly pure isoprene composition useful in the invention has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene. In particular embodiments, the composition also has greater than about 2 mg of isoprene.

In some embodiments, the method for producing a fuel constituent from isoprene comprises obtaining a commercially beneficial amount of highly pure isoprene composition. In some embodiments, the highly pure isoprene composition useful in the invention has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms. In some embodiments, the highly pure isoprene composition has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of ethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more of the foregoing. In particular embodiments, the highly pure isoprene composition has greater than about 2 mg of isoprene and has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.

In some embodiments, the method for producing a fuel constituent from isoprene comprises obtaining a commercially beneficial amount of highly pure isoprene composition. In some embodiments, the highly pure isoprene composition useful in the invention includes isoprene and one or more second compounds selected from the group consisting of 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. In various embodiments, the amount of one of these second components relative to the amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is at greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w). In some embodiments, the amount of methanethiol relative to the amount of isoprene in units of percentage by weight is at less than 0.01% (w/w).

In some embodiments, the method for producing a fuel constituent from isoprene comprises obtaining a commercially beneficial amount of highly pure isoprene composition. In some embodiments, the method comprises obtaining a commercially beneficial amount of highly pure isoprene composition from a biological process comprising: (a) culturing cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide under suitable culture conditions for the production of isoprene, wherein the cells (i) produce greater than about 400 nmole/g_(wcm)/hr of isoprene, (ii) convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, or (iii) have an average volumetric productivity of isoprene greater than about 0.1 mg/L_(broth)/hr of isoprene, and (b) producing isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide, e.g. a naturally-occurring polypeptide from a plant such as Pueraria, and (ii) is operably linked to a promoter, e.g. a T7 promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions. In some embodiment, the cells further comprise a heterologous nucleic acid encoding an IDI polypeptide. In some embodiments, the cells further comprise a heterologous nucleic acid encoding an MDV pathway polypeptide. In some embodiment, the method comprises producing isoprene using the methods described in U.S. provisional patent application Nos. 61/134,094, filed on Jul. 2, 2008, WO 2010/003007, and U.S. patent application Ser. No. 12/335,071, filed Dec. 15, 2008 (US 2009/0203102 A1), which are incorporated by reference in their entireties.

In some embodiment, the method comprises obtaining a gas phase (off-gas) produced by cells in culture that produces isoprene. In some embodiments, the cells in culturing produce greater than about 400 nmole/g_(wcm)/hr of isoprene. In some embodiments, the gas phase comprises greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg/L of isoprene when sparged at a rate of 1 vvm. In some embodiments, the volatile organic fraction of the gas phase comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In some embodiments, the volatile organic fraction of the gas phase comprises less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In some embodiments, the volatile organic fraction of the gas phase has less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In particular embodiments, the volatile organic fraction of the gas phase has greater than about 2 mg of isoprene and has greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction.

In some embodiment, the method comprises obtaining a gas phase (off-gas) produced by cells in culture that produces isoprene. In some embodiments, the volatile organic fraction of the gas phase has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a compound that inhibits the polymerization of isoprene for any compound in the volatile organic fraction of the gas phase that inhibits the polymerization of isoprene. In particular embodiments, the volatile organic fraction of the gas phase also has greater than about 2 mg of isoprene. In some embodiments, the volatile organic fraction of the gas phase has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms. In some embodiments, the volatile organic fraction of the gas phase has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of ethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more of the foregoing. In particular embodiments, the volatile organic fraction of the gas phase has greater than about 2 mg of isoprene and has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms. In some embodiments, the volatile organic fraction of the gas phase has includes isoprene and one or more second compounds selected from the group consisting of 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. In various embodiments, the amount of one of these second components relative to amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is at greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w) in the volatile organic fraction of the gas phase. In some embodiments, the method further includes recovering the isoprene from the gas phase. For example, the isoprene in the gas phase comprising isoprene can be recovered using standard techniques, such as gas stripping, membrane enhanced separation, fractionation, adsorption/desorption, pervaporation, thermal or vacuum desorption of isoprene from a solid phase, or extraction of isoprene immobilized or absorbed to a solid phase with a solvent (see, for example, U.S. Pat. Nos. 4,703,007 and 4,570,029, which are each hereby incorporated by reference in their entireties, particularly with respect to isoprene recovery and purification methods). In some particular embodiments, extractive distillation with an alcohol (such as ethanol, methanol, propanol, or a combination thereof) is used to recover the isoprene. In some embodiments, the recovery of isoprene involves the isolation of isoprene in a liquid form (such as a neat solution of isoprene or a solution of isoprene in a solvent). Gas stripping involves the removal of isoprene vapor from the fermentation off-gas stream in a continuous manner. Such removal can be achieved in several different ways including, but not limited to, adsorption to a solid phase, partition into a liquid phase, or direct condensation (such as condensation due to exposure to a condensation coil or do to an increase in pressure). In some embodiments, membrane enrichment of a dilute isoprene vapor stream above the dew point of the vapor resulting in the condensation of liquid isoprene. In some embodiments, the recovered isoprene is compressed and condensed.

The recovery of isoprene may involve one step or multiple steps. In some embodiments, the removal of isoprene vapor from a fermentation off-gas and the conversion of isoprene to a liquid phase are performed simultaneously. For example, isoprene can be directly condensed from the off-gas stream to form a liquid. In some embodiments, the removal of isoprene vapor from the fermentation off-gas and the conversion of isoprene to a liquid phase are performed sequentially. For example, isoprene may be adsorbed to a solid phase and then extracted from the solid phase with a solvent.

In some embodiments, a method is provided for producing a cyclic dimer of isoprene comprising heating neat bioisoprene. The cyclic dimers of isoprene produced may be 6-membered ring dimers (e.g. a [2+4] electrocyclization product such as limonene) or 8-membered ring dimers or a mixture thereof. Examples of 6-membered ring dimers include but not limited to 1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene, 1-methyl-5-(prop-1-en-2-yl)cyclohex-1-ene, 1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene and the like. Examples of 8-membered ring dimers include but not limited to 1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta-1,5-diene and the like. In one embodiment, neat biologically produced isoprene is dimerized by heating to a temperature of about 100° C. to about 300° C., preferably about 150° C. to about 250° C. The pressure is maintained at about 2 to 3 atm. In another embodiment, a catalyst suitable for catalyzing dimerization of conjugated dienes may be used. The proportions of various dimers in the product mixture may be controlled by the catalyst and other reactions conditions. For example, a nickel catalyst can promote formation of 8-membered ring dimers.

In some embodiments, a method is provided for producing a cyclic dimer of isoprene comprising photo-dimerization of bioisoprene. The cyclic dimer of isoprene produced may be one or more 4-membered ring dimer or a mixture thereof. Examples of Examples of 4-membered ring dimers include but not limited to 1,2-di(prop-1-en-2-yl)cyclobutane, 1,3-di(prop-1-en-2-yl)cyclobutane, 1-methyl-1-vinyl-3-(prop-1-en-2-yl)cyclobutane, 1-methyl-1-vinyl-2-(prop-1-en-2-yl)cyclobutane, 1,3-dimethyl-1,3-divinylcyclobutane, 1,2-dimethyl-1,2-divinylcyclobutane and the like. In one embodiment, neat biologically produced isoprene is dimerized by irradiating with UV light, preferably in presence of a photosensitizer such as benzophenone.

Isoprene dimers can be hydrogenated to form saturated C₁₀ hydrocarbons that can serve as fuels or blended into fuels. In one embodiment, the unsaturated isoprene dimers are subjected to catalytic hydrogenation to produce partially hydrogenated and/or fully hydrogenated products. Examples of fully hydrogenated products include but not limited to 1-methyl-4-isopropylcyclohexane and 1-methyl-5-isopropylcyclohexane, 1,4-dimethyl-4-ethylcyclohexane, 2,4-dimethyl-4-ethylcyclohexane, 1,2-diisopropylcyclobutane and 1,3-diisopropylcyclobutane, 1-methyl-1-vinyl-3-(prop-1-en-2-yl)cyclobutane, 1-methyl-1-ethyl-2-(prop-2-yl)cyclobutane, 1,3-dimethyl-1,3-ethylcyclobutane, 1,2-dimethyl-1,2-diethylcyclobutane, 1,5-dimethylcyclooctane, 1,6-dimethylcyclooctane and the like.

In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) chemically transforming at least a portion of the highly pure isoprene starting composition to a fuel constituent comprising: (i) heating the highly pure isoprene composition to over or about 100° C. under pressure; (ii) converting at least a portion of the starting isoprene composition to unsaturated cyclic isoprene dimers; and (iii) hydrogenating the unsaturated cyclic isoprene dimers to produce saturated cyclic isoprene dimers. In some embodiments, the starting isoprene composition is heated to over or about 100, 125, 150, 175, 200, 225 or 250° C. under pressure. In some embodiments, the starting isoprene composition is heated in the presence of an antioxidant (e.g. 2,6-di-tert-butyl-4-methylphenol) to prevent radical-mediated polymerization. In one embodiment, the thermal dimerization of isoprene is performed in presence of dinitrocresol as polymerization inhibitor. Suitable antioxidants may be used as polymerization inhibitors. In some embodiments, at least a portion of the starting isoprene composition to a mixture of unsaturated dimers that includes 6 and 8-membered rings, for examples, limonene (1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene), 1-methyl-5-(prop-1-en-2-yl)cyclohex-1-ene, 1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene, 1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta-1,5-diene, 2,6-dimethyl-1,3-cyclooctadiene, 2,6-dimethyl-1,4-cyclooctadiene, 3,7-dimethyl-1,5-cyclooctadiene, 3,7-dimethyl-1,3-cyclooctadiene and 3,6-dimethyl-1,3-cyclooctadiene. In some embodiments, greater than or about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% of isoprene in starting isoprene composition is converted to unsaturated cyclic isoprene dimers. In some embodiments, hydrogenation of the unsaturated cyclic isoprene dimers is catalyzed by a hydrogenation catalyst such as a palladium based catalyst (e.g. Pd/C, e.g. 5% (wt.) Pd/C.) In some embodiments, the saturated cyclic isoprene dimers comprise one or more C10 hydrocarbons selected from the group consisting of 1-methyl-4-isopropylcyclohexane, 1-methyl-3-isopropylcyclohexane, 1,5-dimethylcyclooctane and 1,4-dimethylcyclooctane. In some preferred embodiments, the starting isoprene composition is a bioisoprene composition.

In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) chemically transforming at least a portion of the highly pure isoprene starting composition to a fuel constituent comprising: (i) contacting the highly pure isoprene composition with a catalyst system; (ii) converting at least a portion of the starting isoprene composition to unsaturated isoprene dimers and/or trimers; and (iii) hydrogenating the unsaturated dimers and/or trimers to produce saturated C10 and/or C15 hydrocarbons. In some embodiments, greater than or about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% of isoprene in starting isoprene composition is converted to unsaturated dimers and/or trimers of isoprene. In some embodiments, the starting isoprene composition is contacted with appropriate catalyst systems known in the art to yield one or more of unsaturated cyclic isoprene dimers selected from the group consisting of 1,2-di(prop-1-en-2-yl)cyclobutane, 1,3-di(prop-1-en-2-yl)cyclobutane, 1-methyl-1-vinyl-3-(prop-1-en-2-yl)cyclobutane, 1-methyl-1-vinyl-2-(prop-1-en-2-yl)cyclobutane, 1,3-dimethyl-1,3-divinylcyclobutane, 1,2-dimethyl-1,2-divinylcyclobutane, 1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene), 1-methyl-5-(prop-1-en-2-yl)cyclohex-1-ene, 1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene, 1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta-1,5-diene, 1,4-dimethyl-4-vinyl-1-cyclohexene, 2,4-dimethyl-4-vinyl-1-cyclohexene, 2,7-dimethyl-1,3,7-octatriene, 2,7-dimethyl-2,4,6-octatriene, 2,6-dimethyl-1,3-cyclooctadiene, 2,6-dimethyl-1,4-cyclooctadiene, 3,7-dimethyl-1,5-cyclooctadiene, 3,7-dimethyl-1,3-cyclooctadiene and 3,6-dimethyl-1,3-cyclooctadiene. In some embodiments, the starting isoprene composition is contacted with appropriate catalyst systems known in the art to yield one or more of unsaturated cyclic isoprene trimers such as trimethylcyclododecatrienes and trimethyldodecatetraenes and the like. In some embodiments, the starting isoprene composition is contacted with appropriate catalyst systems known in the art to yield one or more of unsaturated linear dimers and/or trimers of isoprene such as dimethyloctatrienes (e.g. 2,7-dimethyl-1,3,7-octatriene and 2,7-dimethyl-2,4,6-octatriene) and trimethyldodecatetraenes (e.g. 2,6,10-trimethyl-1,5,9,11-dodecatetraene, a-farnesene and β-farnesene) and the like. In some preferred embodiments, the starting isoprene composition is a bioisoprene composition.

In some embodiments, the method for producing a fuel constituent from isoprene comprises contacting the highly pure isoprene composition with a catalyst system to convert at least a portion of the starting isoprene composition to unsaturated isoprene dimers and/or trimers. In some embodiments, the catalyst system comprise a Ni, Fe or Co catalyst and the isoprene in the starting composition is converted an 8-membered ring isoprene dimer comprising dimethylcyclooctadienes such as 1,5-dimethylcycloocta-1,5-diene and 1,6-dimethylcycloocta-1,5-diene. In some embodiments, the catalyst system comprises a nickel compound. Ni-catalyzed dimerization of isoprene yields a dimethyl-1,5-cyclooctadiene mixture consisting of about 90 to 10, 80 to 20, 70 to 30, 60 to 40, 50 to 50, 40 to 60, 30 to 70, 20 to 80 and 10 to 90 ratio of 1,5-dimethyl-1,5-cyclooctadiene to 1,6-dimethyl-1,5-cyclooctadiene. In one embodiment, the catalyst system comprises a 3-component catalyst containing Ni carboxylates or β-ketones, organoaluminum or organomagnesium compounds and substituted triphenylphosphite. For example, a degassed solution of a highly pure isoprene starting composition in anhydrous toluene is mixed with Ni(acac)₂, Et₃Al, and tris(3,4-bis(dimethylamino)phenyl)phosphite under nitrogen atmosphere and the mixture is heated at 95° C. to give dimethylcyclooctadiene. In another embodiment, the catalyst system comprises Fe carboxylates or β-diketone compounds, organo-Al or Mg compounds, and 2,2′-dipyridyl derivatives having electron-donating groups. For example, a starting isoprene composition is heated with a mixture of Fe acetylacetonate, 4,4′-dimethyl-2,2′-dipyridyl, and Et₃Al in toluene to gave 1,6-dimethyl-1,5-cyclooctadiene in good yields. In some preferred embodiments, the starting isoprene composition is a bioisoprene composition.

In some embodiments, the method for producing a fuel constituent from isoprene comprises contacting the highly pure isoprene composition with a catalyst system to convert at least a portion of the starting isoprene composition to unsaturated isoprene dimers and/or trimers. In some embodiments, the catalyst system comprises a Ru, Fe, Ni or Pd catalyst or a combination thereof and the isoprene in the starting composition is converted to a mixture comprising 6-membered ring isoprene dimers. In one embodiment, the catalyst system is a Fe(NO)₂C1-bis(1,5-cyclooctadiene)nickel catalyst system and the isoprene in the starting composition is cyclodimerized at low temperatures (e.g. from −5 to +20° C.) to give 1-methyl- and 2-methyl-4-isopropenyl-1-cyclohexene and 1,4- and 2,4-dimethyl-4-vinyl-1-cyclohexene. In another embodiment, contacting with a mixed catalyst of a tris(substituted hydrocarbyl) phosphite, arsenite, or antimonite and a Group VIII metal(0) compound (e.g. Ni acetylacetonate) converts the isoprene in the starting composition to 1,4-dimethyl-4-vinyl-1-cyclohexene and 1,5-dimethyl-1,5-cyclooctadiene. In another embodiment, contacting an isoprene starting composition with a ruthenium catalyst (e.g. see Itoh, Kenji; Masuda, Katsuyuki; Fukahori, Takahiko; Nakano, Katsumasa; Aoki, Katsuyuki; Nagashima, Hideo, Organometallics (1994), 13(3), 1020-9) converts isoprene to C10 cyclic dimers (e.g. a mixture of dimethyl-cyclooctadienes). The ruthenium catalyst can be synthesized in two steps from RuCl₃ and pentamethylcyclopentadiene (C₅Me₅H). The process may be performed in batch mode and the catalyst recovered and recycled. In some preferred embodiments, the starting isoprene composition is a bioisoprene composition. One embodiment of the method is illustrated in Scheme XI.

In some embodiments, the method for producing a fuel constituent from isoprene comprises contacting the highly pure isoprene composition with a catalyst system to convert at least a portion of the starting isoprene composition to unsaturated isoprene dimers and/or trimers. In some embodiments, the catalyst system comprises a Ni, Ti, Al, or Mg catalyst or a mixture thereof and the isoprene in the starting composition is converted to a mixture comprising cyclic isoprene trimers such as trimethylcyclododecatrienes. In one embodiment, the catalyst system for the trimerization of isoprene comprising Ni and/or Ti, one or more organometallic compound, and a Group VA compound. The reaction may be conducted in a hydroxyl group-containing solvent. In another embodiment, oligomerization of isoprene catalyzed by nickel naphthenate and isoprenemagnesium in the presence of various phosphites as electron donors give cyclic dimers containing dimethylcyclooctadiene, in particular 1,1,1-tris(hydroxymethyl)propane phosphite gives trimethylcyclododecatriene selectively.

In some embodiments, the method for producing a fuel constituent from isoprene comprises contacting the highly pure isoprene composition with a catalyst system to convert at least a portion of the starting isoprene composition to unsaturated isoprene dimers and/or trimers. In some embodiments, the catalyst system comprises a Ni, Pd or Cr catalyst or a mixture thereof and the isoprene in the starting composition is converted telomers or linear dimers and/or trimers. In one embodiment, a catalyst system comprising Pd(0) complexes (e.g. Pd(acac)₂-Ph₃P and Pd(OAc)₂-Ph₃P) catalyzes dimerization and telomerization of isoprene to give linear isoprene dimers (e.g. 2,7-dimethyl-1,3,7-octatriene). In one variation, the reaction is performed in methanol as a solvent and methyl ethers such as methoxydimethyloctadienes (e.g. 1-methoxy-2,7-dimethyl-2,7-octadiene and 3-methoxy-2,7-dimethyl-1,7-octadiene) are produced. In another embodiment, the catalyst system comprises divalent and trivalent transition metal-exchanged montmorillonites (e.g. Cr³⁺-montmorillonite). In another embodiment, the catalyst system comprises a Ni(0)-aminophosphinite catalyst and the isoprene in the starting composition is converted to regioselective tail-to-tail linear dimers. In some variations, the linear dimer formation is accompanied by a competitive cyclodimerization reaction. In another embodiment, the catalyst system comprises a chromium N,N-bis(diarylphosphino)amine catalyst (e.g. see Bowen, L.; Charernsuk, M.; Wass, D. F. Chem. Commun. (2007) 2835-2837) and the isoprene is converted to linear and cyclic C15 trimers. In one variation, the chromium catalyst is made in situ from CrCl₃(THF)₃ and a PNP phosphine ligand.

In some embodiments, the method for producing a fuel constituent from a bioisoprene composition comprises chemically transforming a substantial portion of the isoprene in the bioisoprene composition by (i) contacting the bioisoprene composition with a catalyst for catalyzing trimerization of isoprene to produce an unsaturated isoprene trimer and (ii) hydrogenating the isoprene trimer to produce a fuel constituent. In some embodiments, at least about 95% of isoprene in the bioisoprene composition is converted to non-isoprene compounds during the chemical transformation.

In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) converting at least a portion of the starting isoprene composition to unsaturated isoprene derivatives; and (c) hydrogenating the unsaturated isoprene derivatives to produce saturated compounds. In some embodiments, hydrogenation of unsaturated isoprene derivatives to saturated compounds is performed in batch mode. In some embodiments, hydrogenation of unsaturated isoprene derivatives to saturated compounds is performed in continuous mode. One embodiment of the method is illustrated in Scheme XII.

In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) converting at least a portion of the starting isoprene composition to oxygenated isoprene derivatives; and optionally (c) hydrogenating any unsaturated oxygenated isoprene derivatives to produce saturated oxygenates. In some embodiments, the isoprene starting composition is contacted with a catalyst in the presence of alcohols (e.g. methanol or ethanol or mixtures thereof) to form oxygenated isoprene derivatives comprise one or more unsaturated or saturated ethers (e.g. methyl ethers or ethyl ethers or mixtures thereof). In some embodiments, the catalyst is an acid catalyst such as sulfuric acid, solid phase sulfuric acid (e.g., Dowex Marathon®), liquid and solid-phase fluorosulfonic acids (e.g. trifluoromethanesulfonic acid and Nafion-H (DuPont)). Zeolite catalysts can also been used, for example beta-zeolite, under conditions similar to those described by Hensel et al. [Hensen, K.; Mahaim, C.; Holderich, W. F., Applied Catalysis A: General (1997) 140(2), 311-329.] for the methoxylation of limonene and related monoterpenes. In some embodiments, the isoprene starting compositions or the unsaturated isoprene dimers or trimers undergo oxidation/hydrogenation to form the hydroxylated isoprene derivatives comprise one or more alcohols such as C5, C10 and C15 alcohols and diols. In some embodiments, the isoprene starting composition undergoes hydroxylation/esterification to form alcohols and esters. For example, bioisoprene or an unsaturated intermediate undergoes peroxidation to epoxides with peracids such as peracetic and 3-chloroperbenzoic acid; and hydration to give alcohols and diols with i) water and acid catalysts and ii) hydroboration methods. Such reactions are described in, for example, Michael B. Smith and Jerry March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Sixth Edition, John Wiley & Sons, 2007. In some preferred embodiments, the starting isoprene composition is a bioisoprene composition.

In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) partially hydrogenating the starting isoprene composition to mono-olefins (e.g. 2-methylbut-1-ene, 3-methyl-but-1-ene and 2-methylbut-2-ene); and (c) producing the fuel constituent from the mono-olefins. In one embodiment, the mono-olefin undergoes dimerization. In another embodiment, the mono-olefin is reacted with other olefins using traditional hydrocarbon cationic catalysis, such as that used to covert isobutylene to isooctane. The product of dimerization or reaction with other olefins are then undergoes hydrogenation to give saturated fuel constituents. In some preferred embodiments, the highly pure isoprene is a bioisoprene composition. In some embodiment, the starting isoprene composition partially hydrogenated using a palladium catalyst, such as Pd/CaCO₃, Pd/BaSO₄, Pd/C, Pd black, Pd/SiO₃, Pd/Al₂O₃, or Pd/SiO₂. In some embodiment, the starting isoprene composition partially hydrogenated using a silica-supported polyamidoamine (PAMAM) dendrimer-palladium complex. In some embodiment, the starting isoprene composition partially hydrogenated using a palladium-gold and palladium-silver catalysts (e.g., Pd—Au/SiO₂ and Pd—Ag/SiO₂) have high selectivity for reducing isoprene to mono-olefins over fully reduced C5 alkanes. In some embodiment, the starting isoprene composition partially hydrogenated using a Group VIB metal on an inorganic support (e.g., a metal zeolite) as a catalyst, e.g. a Mo/Al₂O₃ catalyst. In some embodiment, the starting isoprene composition partially hydrogenated using a monolithic catalyst bed, which may be in a honeycomb configuration. Catalyst support materials for the monolithic catalyst bed may include metals such as nickel, platinum, palladium, rhodium, ruthenium, silver, iron, copper, cobalt, chromium, iridium, tin, and alloys or mixtures thereof. In some embodiment, the starting isoprene composition partially hydrogenated using an eggshell Pd/d-Al₂O₃ catalyst, particularly if the reaction is free of water. In some embodiment, the starting isoprene composition partially hydrogenated using a Group VIII metal catalyst promoted by a metal from Group IB, VIIB, VIIB, or zinc to reduce poisoning of the catalyst. Methods for converting isoprene into isoamylenes (mono-olefins) can be performed in continuous or batch mode using both homogeneous or heterogeneous catalysts. One embodiment of the method is illustrated in Scheme XIII.

In some embodiments, isoamylenes produce from partial hydrogenatin of isoprene undergos trimerization in liquid phase over ion exchange resins and zeolites. See, GranGranollers, Ind. Eng. Chem. Res., 49 (8), pp 3561-3570 (2001).

In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) partially hydrogenating the starting isoprene composition to a mono-olefin (e.g. 2-methylbut-1-ene, 3-methyl-but-1-ene and 2-methylbut-2-ene); (c) contacting the mono-olefin with a catalyst for dimerization of mono-olefins to form a dimate with another mono-olefin; and (d) hydrogenating the product of dimerization (dimate) to produce saturated hydrocarbon fuel constituents. In some preferred embodiments, the highly pure isoprene is a bioisoprene composition. In some embodiments, the other mono-olefin is an isoamylene derived from the highly pure isoprene starting composition. In some embodiments, the other mono-olefin is an olefin from another source such as propylene, butane or isobutene. In some embodiments, the catalyst for dimerization is a resin catalyst (e.g., Amberlyst, Amberlyst 35, Amberlyst 15, Amberlyst XN1010, Amberlyst XE586). In some embodiments, the catalyst for dimerization is a catalytic material containing an acidic mesoporous molecular sieve, such as a mesoporous sieve embedded in a zeolite structure, ZSM-5, ferrierite, ZSM-22, or ZSM-23. In some embodiments, the catalyst for dimerization is the catalytic material is thermally stable at high temperatures (e.g., at least 900° C.). In some embodiments, the catalyst for dimerization is a catalytic material containing an acidic mesoporous molecular sieve. In some embodiments, the catalyst for dimerization is a solid acidic catalyst (e.g., a solid phosphoric acid catalyst, acidic ion exchange resins). One embodiment of the method is illustrated in Scheme XIV.

In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) partially hydrogenating the starting isoprene composition to an isoamylene (e.g. 2-methylbut-1-ene, 3-methyl-but-1-ene and 2-methylbut-2-ene); (c) contacting the isoamylene with an alcohol and a catalyst to form an oxygenate; and (c) producing producing the fuel constituent from the isoamylene oxygenate. In some preferred embodiments, the highly pure isoprene is a bioisoprene composition. In some embodiments, the isoamylene is contacted with ethanol and the fuel oxygenate formed is an ether such as TAME.

In some embodiments, any of the methods described herein further include purifying the starting isoprene composition before the chemical transformation. For example, the isoprene produced using the compositions and methods of the invention can be purified using standard techniques. Purification refers to a process through which isoprene is separated from one or more components that are present when the isoprene is produced. In some embodiments, the isoprene is obtained as a substantially pure liquid. Examples of purification methods include (i) distillation from a solution in a liquid extractant and (ii) chromatography. As used herein, “purified isoprene” means isoprene that has been separated from one or more components that are present when the isoprene is produced. In some embodiments, the isoprene is at least about 20%, by weight, free from other components that are present when the isoprene is produced. In various embodiments, the isoprene is at least or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%, by weight, pure. Purity can be assayed by any appropriate method, e.g., by column chromatography, HPLC analysis, or GC-MS analysis. In some embodiments, bioisoprene is recovered from fermentation off-gas by initial adsorption to activated carbon, followed by desorption and condensation to give a liquid isoprene composition for chemical transformation to a fuel constituent.

Continuous Process for Producing Fuels

The invention also provides a continuous process for producing a fuel constituent from isoprene comprising: (a) continuously producing a commercially beneficial amount of highly pure isoprene; and (b) continuously transforming chemically at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent. In a continuous process according to the invention, a stream of highly pure isoprene is produced continuously, for example, by a biological process (e.g. culturing cells producing isoprene) and is passed to a reactor for chemical transformation. In some embodiments, the isoprene from a fermentation off-gas is separated from water and other permanent gases (e.g., O₂, N₂ and CO₂) using extractive distillation, condensation, adsorption or membranes in a continuous purification unit, where the oxygen level is lowered to ppm range and other impurities of concern are removed; the isoprene vapor is catalytically converted to dimers (C10) and trimers (C15) using a heterogeneous catalyst; the desired product is separated and the unreacted isoprene is returned to the reactor for further conversion. In some embodiments, the catalytic dimerization is monitored by measuring the levels of desirable C10 hydrocarbons in the gas phase.

In some embodiments, a stream of biologically produced isoprene is passed through a chemical reactor (e.g., a reaction tube) maintained at a temperature of about 100° C. to about 300° C., where the biologically produced isoprene undergoes dimerization reactions. In one variation, the isoprene dimers are separated from the product stream and at least a portion of the unreacted isoprene is recycled back into the chemical reactor. In one embodiment, the unsaturated isoprene dimers produced is contacted with a hydrogenation catalyst and a hydrogen source in a second chemical reactor producing partially hydrogenated and/or fully hydrogenated products. The process can further comprise a step of recovering isoprene from the off-gas of a bio-reactor producing isoprene.

In some embodiments, a stream of bioisoprene containing co-produced hydrogen is first separated from the hydrogen gas, the bioisoprene stream is passed through a chemical reactor (e.g., a reaction tube) maintained at a temperature of about 100° C. to about 300° C., where the biologically produced isoprene undergoes dimerization reactions. In one variation, the isoprene dimers are separated from the product stream and at least a portion of the unreacted isoprene is recycled back into the chemical reactor. In one embodiment, the unsaturated isoprene dimers produced is contacted with a hydrogenation catalyst and a hydrogen source in a second chemical reactor producing partially hydrogenated and/or fully hydrogenated products. In one variation, the hydrogen stream isolated from the isoprene-hydrogen co-production is used in the hydrogenation step. The process can further comprise a step of recovering isoprene from the off-gas of a bio-reactor producing isoprene. The process can further comprise a step of purifying the hydrogen stream isolated from the isoprene-hydrogen co-production using purification methods known in the art such as cryogenic condensation and solid matrix adsorption.

Removal of Dienes and Polymers from Fuel Products

Fuel compositions often contain unsaturated compounds (olefins, diolefins and polyolefins) that can form gums, resins, polymers and other undesirable byproducts over time (for example, see Pereira and Pasa (2006) Fuel, 85, 1860-1865 and references therein). In general, as the degree of unsaturation increases of a given compound, the more likely that compound is to form such byproducts. Isoprene is a 1,3-diene that readily forms undesirable polymeric byproducts when present in fuel compositions. While there exist fuel additives (anti-oxidants, radical quenchers etc.) that can reduce the extent of byproduct formation, such byproducts can still form over time. Olefins can also contribute to the formation of ground-level ozone when released into the atmosphere upon evaporation from fuels, or as the result of incomplete combustion of olefin-containing fuels.

Accordingly, fuel compositions derived wholly or in part from isoprene should contain little to no free isoprene. A range of methods can be used to either remove isoprene from fuel compositions such as purification by distillation, reaction with alcohols to form ethers, or hydrogenation to convert isoprene to saturated derivatives. Alternately, isoprene can be treated with a dienophile such as malic anhydride producing inert adducts that do not contribute to the formation of undesirable byproducts.

Provided are fuel compositions comprising isoprene derivatives that are substantially free of isoprene. In some embodiments, the fuel composition contains less than 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10% or 15% isoprene. Also provided are methods for chemically transforming a bioisoprene composition to fuel compositions comprising isoprene derivatives that are substantially free of isoprene. In some embodiments, a substantial portion of the isoprene from the bioisoprene composition is converted to fuel constituents. In some embodiments, a substantial portion of the isoprene from the bioisoprene composition is converted to intermediates that can be further converted to produce fuel constituents. In some embodiments, a substantial portion of the isoprene from the bioisoprene composition is converted to compounds other than isoprene. A “substantial portion” is 99.99%, 99.9%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80%.

In some instances, isoprene and other conjugated dienes can form polymeric products with gum-like consistencies that can reduce the yields of desired products and/or deactivate catalysts. (See, e.g., R. C. C. Pereira and V. M. D. Pasa. Fuel 85 (2006) 1860-1865.) In some embodiments, methods are provided for determining the amount of conjugated diene present in a product mixture. D. F. Andrade et al. describe various methods for determining the amount of conjugated diene present (Fuel (2010), doi:10.106/j.fuel/2010.01.003), including the following: 1) UOP-326 method (maleic anhydride method), a semi-quantitative method in which the amount of maleic anhydride consumed via Diels-Alder reaction with the diene is measured; 2) polarography; 3) gas chromatography, in which the diene may be reacted with a derivatization agent, such as 4-methyl-1,2,4-triazoline-3,5-dione (MTAD) or 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); 4) HPLC; 5) supercritical fluid chromatography; 6) NMR; 7) UV and near IR spectroscopy; and 8) other spectroscopic methods, which may include first derivatizing the diene with p-nitrobenezenediazonium fluoroborate.

Method are provided herein for minimizing gum-formation and/or reducing the amount of gum that has been produced. In some embodiments, an anti-oxidant is used to minimize gum-formation. In other embodiments, polymeric by-products are recycled back to the process stream. In some embodiments, depolymerization of polymeric by-products is carried out via olefin metathesis. Olefin metathesis can be carried out using a molybdenum or tungsten catalyst, such as 2,6-diisopropylphenylimido neophylidene molybdenum bis(2-tertbutylphenoxide). (See U.S. Pat. No. 5,446,102.)

In some embodiments, any of the methods described herein further include characterizing the products of these reactions and assessing the potential fuel value. For example, the products are characterized by standard methods known in the art, e.g. GC/MS, NMR, UV-Vis and IR spectroscopies, boiling temperature, density and other physical properties. The products can be further characterized by dual carbon-isotopic fingerprinting (see, U.S. Pat. No. 7,169,588). The potential fuel value of the products are assessed by parameters measuring fuel properties such as the energy density, heating value, water solubility, octane/cetane number, density, viscosity, surface tension, enthalpy of vaporization, vapor diffusivity, flash point, autoignition point, flammability limits, cloud point and chemical stability.

As with commercial petroleum fuels, BioIsoFuel™ products can be tested for their acidity, density, trace mineral content, benzene, total aromatics content, water content, and corrosivity. To assure that minor impurities in BioIsoFuel™ products are not adversely affecting their material properties, samples can also be tested for their corrosivity and compatibility with fuel systems by standard ASTM tests such as Karl Fischer for water and copper strip for corrosivity.

Fuel Compositions

The invention provides a fuel composition comprising a fuel constituent produced by any of the methods and processes described herein. In some embodiments, the fuel constituent comprises one or more of the hydrocarbons selected from the group consisting of saturated C10 and C₁₋₅ hydrocarbons derived from isoprene and oxygenated derivatives of isoprene. The hydrocarbon BioIsoFuel™ products are expected to be completely compatible with petroleum gasoline and diesel. Blends of BioIsoFuel™ with commercial petroleum fuels are expected to have more desirable properties, as they do not contain acids, sulfur, aromatics, or other undesirable impurities.

In some preferred embodiments, the fuel constituent comprises derivatives of bioisoprene. It is anticipated that there will be a significant demand for fuels derived from bioisoprene which is made from renewable, non-petrochemical based resources. It is believed that industrial customers and consumers would prefer to purchase fuel components derived from such environmentally friendly sources to those that are made with isoprene derived from a petrochemical process. It is further believed that customers would be willing to pay premium prices for such environmentally friendly products that are made with renewable resources. Fuel components derived from bioisoprene compositions described herein offer the benefit of being verifiable as to be derived from non-petrochemical based resources.

In some embodiments, the fuel constituent comprises one or more of the saturated cyclic C10 hydrocarbons such as 1,2-bis(isopropyl)cyclobutane, 1,2-bis(isopropyl)cyclobutane, 1-methyl-4-isopropylcyclohexane, 1-methyl-3-isopropylcyclohexane, 1-ethyl-1,4-dimethylcyclohexane, 1-ethyl-1,3-dimethylcyclohexane, 1,5-dimethylcyclooctane and 1,4-dimethylcyclooctane. In some embodiments, the fuel constituent comprises one or more of the saturated cyclic C15 hydrocarbons such as 1,5,9-trimethylcyclododecane and 1,5,10-trimethylcyclododecane. In some embodiments, the fuel constituent comprises one or more of the saturated aliphatic C10 hydrocarbons such as 2,6-dimethyloctane, 2,7-dimethyloctane and 3,6-dimethyloctane. In some embodiments, the fuel constituent comprises one or more of the saturated aliphatic C15 hydrocarbons such as 2,6,10-trimethyldodecane, 2,7,10-trimethyldodecane and 3,7,10-trimethyldodecane. In some embodiments, the fuel constituent comprises 2-methylbutane. In some embodiments, the fuel constituent comprises one or more of the hydrocarbons selected from the group consisting of 1,2-bis(isopropyl)cyclobutane, 1,2-bis(isopropyl)cyclobutane, 1-methyl-4-isopropylcyclohexane, 1-methyl-3-isopropylcyclohexane, 1-ethyl-1,4-dimethylcyclohexane, 1-ethyl-1,3-dimethylcyclohexane, 1,5-dimethylcyclooctane, 1,4-dimethylcyclooctane, 1,5,9-trimethylcyclododecane, 1,5,10-trimethylcyclododecane, 2,6-dimethyloctane, 2,7-dimethyloctane, 3,6-dimethyloctane, 2,6,10-trimethyldodecane, 2,7,10-trimethyldodecane, 3,7,10-trimethyldodecane and 2-methylbutane.

In some embodiments, the fuel constituent comprises one or more of the saturated hydroxylates such as C5 alcohols and diols (e.g. 3-methyl-butan-1-ol, 2-methyl-butan-1-ol and 2-methyl-butan-2-ol, 3-methyl-butan-1,3-diol and 2-methyl-butan-2,3-diol), C-10 alcohols and diols (e.g. 3,7-dimethyloctan-1-ol, 2,7-dimethyloctan-1-ol, 2,7-dimethyloctan-2-ol and 2,7-dimethyloctan-2,7-diol) and cyclic C-10 alcohols (e.g. 2-(4-methylcyclohexyl)propan-2-ol, 2-(4-methylcyclohexyl)propan-1-ol, 2-(1,4-dimethylcyclohexyl)ethanol and 4-ethyl-1,4-dimethylcyclohexanol). In some embodiments, the fuel constituent comprises one or more of the saturated ethers such as 1,3-diethoxy-3-methylbutane, 1-ethoxy-3-methylbutane, 1-methoxy-2,7-dimethyloctane and 3-methoxy-2,7-dimethyloctane. In some embodiments, the fuel constituent comprises one or more of the isoprene derived oxygenates selected from the group consisting of 3-methyl-butan-1-ol, 2-methyl-butan-1-ol, 2-methyl-butan-2-ol, 3-methyl-butan-1,3-diol, 2-methyl-butan-2,3-diol, 3,7-dimethyloctan-1-ol, 2,7-dimethyloctan-1-ol, 2,7-dimethyloctan-2-ol, 2,7-dimethyloctan-2,7-diol, 2-(4-methylcyclohexyl)propan-2-ol, 2-(4-methylcyclohexyl)propan-1-ol, 2-(1,4-dimethylcyclohexyl)ethanol, 4-ethyl-1,4-dimethylcyclohexanol, 1,3-diethoxy-3-methylbutane, 1-ethoxy-3-methylbutane, 1-methoxy-2,7-dimethyloctane and 3-methoxy-2,7-dimethyloctane.

In some embodiments, the fuel constituent comprises less than or about 0.5 μg/L a product from a C5 hydrocarbon other than isoprene after undergoing the steps according to any of the methods and processes described herein. In one embodiment, the fuel constituent is substantially free of a product from a C5 hydrocarbon other than isoprene after undergoing the steps according to any of the methods and processes described herein. In some embodiments, the fuel constituent comprises less than or about 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the starting composition. In some embodiments, the fuel constituent comprises C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) at a concentration of less than 100, 10, 1, 0.1 or 0.01 ppm.

It is understood that components other than isoprene in the bioisoprene compositions described herein will be converted to different products after undergoing various processes described herein. The sensitivities of metal-based catalysts used in the methods/processes to the components other than isoprene are different depending on the nature and levels of these components. For the thermal dimerization process, a far wider range of compounds will be tolerated. In some cases, a component other than isoprene will react with isoprene to produce adducts, for example the Diels-Alder reactions of methacrolein and methyl vinyl ketone with isoprene to give 6-membered products. The unsaturated adducts can be further hydrogenated to saturated derivatives which are present in the fuel constituents and compositions, e.g. 1,4-dimethyl-1-(hydroxymethyl)cyclohexane and 1-(1-hydroxyethyl)-4-methylcyclohexane (see Scheme XV).

In some embodiments, the fuel constituent comprises a product from a compound selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms after undergoing the steps according to any of the methods and processes described herein. In some embodiments, the fuel constituent comprises one or more products from one or more compounds selected from the group consisting of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine after undergoing the steps according to any of the methods and processes described herein. In some embodiments, the fuel constituent comprises one or more compounds selected from the group consisting of 1,4-dimethyl-1-(hydroxymethyl)cyclohexane, 1-(1-hydroxyethyl)-4-methylcyclohexane, 2-methylbutane, 3-methylpentane, 2-propanol, 2-methyl-1-propanol, 2-butanol, 3-methyl-1-butanol, ethyl acetate and 3-methyl-1-butyl acetate. In some embodiments, the fuel constituent comprises one or more compounds selected from the group consisting of 1,4-dimethyl-1-(hydroxymethyl)cyclohexane, 1-(1-hydroxyethyl)-4-methylcyclohexane, 2-methylbutane, 3-methylpentane, 2-propanol, 2-methyl-1-propanol, 2-butanol, 3-methyl-1-butanol, ethyl acetate and 3-methyl-1-butyl acetate at levels greater than or about 10 ppm, 1 ppm, 100 ppb, 10 ppb or 1 ppb.

In some embodiments, the fuel constituent comprises one or more compounds selected from the group consisting of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine at levels greater than or about 10 ppm, 1 ppm, 100 ppb, 10 ppb or 1 ppb.

Carbon Fingerprinting

BioIsoFuels derived from bioisoprene can be distinguished from fuels derived form petrochemical carbon on the basis of dual carbon-isotopic fingerprinting. Additionally, the specific source of biosourced carbon (e.g. glucose vs. glycerol) can be determined by dual carbon-isotopic fingerprinting (see, U.S. Pat. No. 7,169,588, which is herein incorporated by reference).

This method usefully distinguishes chemically-identical materials, and apportions carbon in products by source (and possibly year) of growth of the biospheric (plant) component. The isotopes, ¹⁴C and ¹³C, bring complementary information to this problem. The radiocarbon dating isotope (¹⁴C), with its nuclear half life of 5730 years, clearly allows one to apportion specimen carbon between fossil (“dead”) and biospheric (“alive”) feedstocks [Currie, L. A. “Source Apportionment of Atmospheric Particles,” Characterization of Environmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc) (1992) 3 74]. The basic assumption in radiocarbon dating is that the constancy of ¹⁴C concentration in the atmosphere leads to the constancy of ¹⁴C in living organisms. When dealing with an isolated sample, the age of a sample can be deduced approximately by the relationship t=(−5730/0.693)ln(A/A_(O)) (Equation 14) where t=age, 5730 years is the half-life of radiocarbon, and A and A_(O) are the specific ¹⁴C activity of the sample and of the modern standard, respectively [Hsieh, Y., Soil Sci. Soc. Am J., 56, 460, (1992)]. However, because of atmospheric nuclear testing since 1950 and the burning of fossil fuel since 1850, ¹⁴C has acquired a second, geochemical time characteristic. Its concentration in atmospheric CO₂—and hence in the living biosphere—approximately doubled at the peak of nuclear testing, in the mid-1960s. It has since been gradually returning to the steady-state cosmogenic (atmospheric) baseline isotope rate (¹⁴C/¹²C) of ca. 1.2×10⁻¹², with an approximate relaxation “half-life” of 7-10 years. (This latter half-life must not be taken literally; rather, one must use the detailed atmospheric nuclear input/decay function to trace the variation of atmospheric and biospheric ¹⁴C since the onset of the nuclear age.) It is this latter biospheric ¹⁴C time characteristic that holds out the promise of annual dating of recent biospheric carbon. ¹⁴C can be measured by accelerator mass spectrometry (AMS), with results given in units of “fraction of modern carbon” (f_(M)). f_(M) is defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the ¹⁴C/¹²C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (plant material), f_(M) ^(˜)1.1.

The stable carbon isotope ratio (¹³C/¹²C) a complementary route to source discrimination and apportionment. The ¹³C/¹²C ratio in a given biosourced material is a consequence of the ¹³C/¹²C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed and also reflects the precise metabolic pathway. Regional variations also occur. Petroleum, C₃ plants (the broadleaf), C₄ plants (the grasses), and marine carbonates all show significant differences in ¹³C/¹²C and the corresponding d¹³C values. Furthermore, lipid matter of C₃ and C₄ plants analyze differently than materials derived from the carbohydrate components of the same plants as a consequence of the metabolic pathway. Within the precision of measurement, ¹³C shows large variations due to isotopic fractionation effects, the most significant of which for the instant invention is the photosynthetic mechanism. The major cause of differences in the carbon isotope ratio in plants is closely associated with differences in the pathway of photosynthetic carbon metabolism in the plants, particularly the reaction occurring during the primary carboxylation, i.e., the initial fixation of atmospheric CO₂. Two large classes of vegetation are those that incorporate the “C₃” (or Calvin-Benson) photosynthetic cycle and those that incorporate the “C₄” (or Hatch-Slack) photosynthetic cycle. C₃ plants, such as hardwoods and conifers, are dominant in the temperate climate zones. In C₃ plants, the primary CO₂ fixation or carboxylation reaction involves the enzyme ribulose-1,5-diphosphate carboxylase and the first stable product is a 3-carbon compound. C₄ plants, on the other hand, include such plants as tropical grasses, corn and sugar cane. In C₄ plants, an additional carboxylation reaction involving another enzyme, phosphoenol-pyruvate carboxylase, is the primary carboxylation reaction. The first stable carbon compound is a 4-carbon acid which is subsequently decarboxylated. The CO₂ thus released is refixed by the C₃ cycle.

Both C₄ and C₃ plants exhibit a range of ¹³C/¹²C isotopic ratios, but typical values are ca. −10 to −14 per mil (C₄) and −21 to −26 per mil (C₃₋) [Weber et al., J. Agric. Food Chem., 45, 2942 (1997)]. Coal and petroleum fall generally in this latter range. The ¹³C measurement scale was originally defined by a zero set by pee dee belemnite (PDB) limestone, where values are given in parts per thousand deviations from this material. The “d¹³C”, values are in parts per thousand (per mil), abbreviated ‰, and are calculated as follows (Equation 15):

$\begin{matrix} {{\delta^{13}C} \equiv {\frac{\left( {}^{13}{C/^{12}C} \right)_{sample} - \left( {}^{13}{C/^{12}C} \right)_{standard}}{\left( {}^{13}{C/^{12}C} \right)_{standard}} \times 100\%}} & \left( {{Equation}\mspace{14mu} 15} \right) \end{matrix}$ Since the PDB reference material (RM) has been exhausted, a series of alternative RMs have been developed in cooperation with the IAEA, USGS, NIST, and other selected international isotope laboratories. Notations for the per mil deviations from PDB is d¹³C. Measurements are made on CO₂ by high precision stable ratio mass spectrometry (IRMS) on molecular ions of masses 44, 45 and 46.

For isoprene derived from extractive distillation of C₅ streams from petroleum refineries, δ¹³C is about −22‰ to about −24‰. This range is typical for light, unsaturated hydrocarbons derived from petroleum, and products derived from petroleum-based isoprene typically contain isoprenic units with the same δ¹³C. Bioisoprene produced by fermentation of corn-derived glucose (δ¹³C −10.73‰) with minimal amounts of other carbon-containing nutrients (e.g., yeast extract) produces isoprene which can be polymerized into polyisoprene with δ¹³C −14.66‰ to −14.85‰. Products produced from such bioisoprene are expected to have δ¹³C values that are less negative than those derived from petroleum-based isoprene. For isoprene derived from the reaction of isobutylene with formaldehyde, δ¹³C values can be about −34.4‰ because formaldehyde is often derived from feedstocks with much more negative δ¹³C values.

The fuels and fuel constituents of this invention which are made with isoprene from the cell cultures that utilize bio-renewable carbon sources can be identified as such by virtue of their d¹³C value and other fuel characteristics. In some embodiments, the fuel constituent derived from bioisoprene has δ¹³C values of greater (less negative) than −22‰. In some embodiments, the fuel constituent derived from bioisoprene has δ¹³C values of greater than −20, −18, −16, −14, −12, or −10‰. In some embodiments, the fuel constituent derived from bioisoprene has a δ¹³C value which is within the range of −22 to −10, −21 to −12, or −20 to −14‰. In some embodiments, the fuel constituent derived from bioisoprene has a δ¹³C value which is within the range of −34 to −24, −34 to −25, −33 to −25, −32 to −24, −32 to −25, −31 to −25, −30 to −29, −30.0 to −29.5, −29.5 to −28.5, or −29.0 to −28.5‰.

In some embodiments, the fuel constituent derived from bioisoprene comprises radioactive carbon-14. In some embodiments, the ¹⁴C/¹²C ratio is greater than or about 1.0×10⁻¹², 1.05×10⁻¹², 1.1×10⁻¹², 1.15×10⁻¹², or 1.2×10⁻¹². In some embodiments, the fuel constituent derived from bioisoprene has an f_(M) value of greater than or about 0.9, 0.95, 1.0, 1.05 or 1.1. In some embodiments, the fuel constituent derived from bioisoprene has an f_(M) value of greater than or about 0.9, 0.95, 1.0, 1.05 or 1.1 and δ¹³C values of greater (less negative) than −22‰. In some embodiments, the fuel constituent derived from bioisoprene has an f_(M) value of greater than or about 0.9, 0.95, 1.0, 1.05 or 1.1 and a δ¹³C value which is within the range of −22 to −10, −21 to −12, or −20 to −14‰. In some embodiments, the fuel constituent derived from bioisoprene has an f_(M) value of greater than or about 0.9, 0.95, 1.0, 1.05 or 1.1 and a δ¹³C value which is within the range of −34 to −24, −34 to −25, −33 to −25, −32 to −24, −32 to −25, −31 to −25, −30 to −29, −30.0 to −29.5, −29.5 to −28.5, or −29.0 to −28.5‰.

The bioisoprene derivatives and the associated BioIsoFuels, intermediates, and mixtures may be completely distinguished from their petrochemical derived counterparts on the basis of ¹⁴C (f_(M)) and dual carbon-isotopic fingerprinting, indicating new compositions of matter.

In some embodiments, the fuel constituent of the invention has an energy density higher than that of ethanol. In some embodiments, the fuel constituent boosts the cetane number of a fuel, e.g., a petroleum-based fuel. In some embodiments, the fuel constituent reduces emission of petroleum based fuels. In some embodiments, the fuel composition has an octane number in the range between about 80 to about 120. In some embodiments, the fuel composition has a cetane number in the range between about 30 to about 130.

In some embodiments, a fuel composition of the invention comprises one or more dimethylcyclooctane compounds. In some embodiments, a fuel composition of the invention comprises one or more C10 hydrocarbons such as substituted cyclohexanes. In some embodiments, a fuel composition of the invention comprises one or more isoprene derived oxygenates described herein. In some embodiments, any of the fuel compositions described herein further comprises a petroleum based fuel in the amount of from about 1% to about 95% by weight or volume, based on the total weight or volume of the total fuel composition.

The invention further provides methods for making a fuel composition comprising obtaining a petroleum distillate and adding a fuel constituent of the invention.

The invention can be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting.

EXAMPLES Example 1 Production of Isoprene in E. coli Expressing Recombinant Kudzu Isoprene Synthase

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthase in E. coli

The protein sequence for the kudzu (Pueraria montana) isoprene synthase gene (IspS) was obtained from GenBank (AAQ84170). A kudzu isoprene synthase gene, optimized for E. coli codon usage, was purchased from DNA2.0 (SEQ ID NO:1). The isoprene synthase gene was removed from the supplied plasmid by restriction endonuclease digestion with BspLU11I/PstI, gel-purified, and ligated into pTrcHis2B (Invitrogen) that had been digested with NcoI/PstI. The construct was designed such that the stop codon in the isoprene synthase gene 5′ to the PstI site. As a result, when the construct was expressed the His-Tag is not attached to the isoprene synthase protein. The resulting plasmid, pTrcKudzu, was verified by sequencing (FIGS. 2 and 3; SEQ ID NO:2).

The isoprene synthase gene was also cloned into pET16b (Novagen). In this case, the isoprene synthase gene was inserted into pET16b such that the recombinant isoprene synthase protein contained the N-terminal His tag. The isoprene synthase gene was amplified from pTrcKudzu by PCR using the primer set pET-His-Kudzu-2F: 5′-CGTGAGATCATATGTGTGCGACCTCTTCTCAATTTAC (SEQ ID NO:49) and pET-His-Kudzu-R: 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:50). These primers added an NdeI site at the 5′-end and a BamH1 site at the 3′ end of the gene respectively. The plasmid pTrcKudzu, described above, was used as template DNA, Herculase polymerase (Stratagene) was used according to manufacture's directions, and primers were added at a concentration of 10 pMols. The PCR was carried out in a total volume of 25 μl. The PCR product was digested with NdeI/BamH1 and cloned into pET16b digested with the same enzymes. The ligation mix was transformed into E. coli Top10 (Invitrogen) and the correct clone selected by sequencing. The resulting plasmid, in which the kudzu isoprene synthase gene was expressed from the T7 promoter, was designated pETNHisKudzu (FIGS. 4 and 5; SEQ ID NO:3).

The kudzu isoprene synthase gene was also cloned into the low copy number plasmid pCL1920. Primers were used to amplify the kudzu isoprene synthase gene from pTrcKudzu described above. The forward primer added a HindIII site and an E. coli consensus RBS to the 5′ end. The PstI cloning site was already present in pTrcKudzu just 3′ of the stop codon so the reverse primer was constructed such that the final PCR product includes the PstI site. The sequences of the primers were: HindIII-rbs-Kudzu F: 5′-CATATGAAAGCTTGTATCGATTAAATAAGGAGGAATAAACC (SEQ ID NO:51) and BamH1-Kudzu R:

5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:50). The PCR product was amplified using Herculase polymerase with primers at a concentration of 10 pmol and with 1 ng of template DNA (pTrcKudzu). The amplification protocol included 30 cycles of (95° C. for 1 minute, 60° C. for 1 minute, 72° C. for 2 minutes). The product was digested with HindIII and PstI and ligated into pCL 1920 which had also been digested with HindIII and PstI. The ligation mix was transformed into E. coli Top10. Several transformants were checked by sequencing. The resulting plasmid was designated pCL-lac-Kudzu (FIGS. 6 and 7; SEQ ID NO:4).

II. Determination of Isoprene Production

For the shake flask cultures, one ml of a culture was transferred from shake flasks to 20 ml CTC headspace vials (Agilent vial cat#5188 2753; cap cat#5188 2759). The cap was screwed on tightly and the vials incubated at the equivalent temperature with shaking at 250 rpm. After 30 minutes the vials were removed from the incubator and analyzed as described below (see Table 1 for some experimental values from this assay).

In cases where isoprene production in fermentors was determined, samples were taken from the off-gas of the fermentor and analyzed directly as described below (see Table 2 for some experimental values from this assay).

The analysis was performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μm film thickness) was used for separation of analytes. The sampler was set up to inject 500 μL, of headspace gas. The GC/MS method utilized helium as the carrier gas at a flow of 1 ml/min. The injection port was held at 250° C. with a split ratio of 50:1. The oven temperature was held at 37° C. for the 2 minute duration of the analysis. The Agilent 5793N mass selective detector was run in single ion monitoring (SIM) mode on m/z 67. The detector was switched off from 1.4 to 1.7 minutes to allow the elution of permanent gases. Under these conditions isoprene (2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. A calibration table was used to quantify the absolute amount of isoprene and was found to be linear from 1 μg/L to 70,000 μg/L. The limit of detection was estimated to be 50 to 100 ng/L using this method.

III. Production of Isoprene in Shake Flasks Containing E. coli Cells Expressing Recombinant Isoprene Synthase

The vectors described above were introduced to E. coli strain BL21 (Novagen) to produce strains BL21/ptrcKudzu, BL21/pCL-lac-Kudzu and BL21/pETHisKudzu. The strains were spread for isolation onto LA (Luria agar)+carbenicillin (50 μg/ml) and incubated overnight at 37° C. Single colonies were inoculated into 250 ml baffled shake flasks containing 20 ml Luria Bertani broth (LB) and carbenicillin (100 μg/ml). Cultures were grown overnight at 20° C. with shaking at 200 rpm. The OD₆₀₀ of the overnight cultures were measured and the cultures were diluted into a 250 ml baffled shake flask containing 30 ml MagicMedia (Invitrogen)+carbenicillin (100 μg/ml) to an OD₆₀₀˜0.05. The culture was incubated at 30° C. with shaking at 200 rpm. When the OD₆₀₀˜0.5-0.8, 400 μM IPTG was added and the cells were incubated for a further 6 hours at 30° C. with shaking at 200 rpm. At 0, 2, 4 and 6 hours after induction with IPTG, 1 ml aliquots of the cultures were collected, the OD₆₀₀ was determined and the amount of isoprene produced was measured as described above. Results are shown in FIG. 8.

IV. Production of Isoprene from BL21/ptrcKudzu in 14 Liter Fermentation

Large scale production of isoprene from E. coli containing the recombinant kudzu isoprene synthase gene was determined from a fed-batch culture. The recipe for the fermentation media (TM2) per liter of fermentation medium was as follows: K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 5 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH₂O. The pH was adjusted to 6.8 with potassium hydroxide (KOH) and q.s. to volume. The final product was filter sterilized with 0.22μ filter (only, do not autoclave). The recipe for 1000× Modified Trace Metal Solution was as follows: Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with a 0.22μ filter.

This experiment was carried out in 14 L bioreactor to monitor isoprene formation from glucose at the desired fermentation, pH 6.7 and temperature 34° C. An inoculum of E. coli strain BL21/ptrcKudzu taken from a frozen vial was prepared in soytone-yeast extract-glucose medium. After the inoculum grew to OD₅₅₀=0.6, two 600 ml flasks were centrifuged and the contents resuspended in 70 ml supernatant to transfer the cell pellet (70 ml of OD 3.1 material) to the bioreactor. At various times after inoculation, samples were removed and the amount of isoprene produced was determined as described above. Results are shown in FIG. 9.

Example 2 Production of Isoprene in E. coli Expressing Recombinant Poplar Isoprene Synthase

The protein sequence for the poplar (Populus alba x Populus tremula) isoprene synthase (Schnitzler, J-P, et al. (2005) Planta 222:777-786) was obtained from GenBank (CAC35696). A gene, codon optimized for E. coli, was purchased from DNA2.0 (p9796-poplar, FIGS. 30 and 31; SEQ ID NO:14). The isoprene synthase gene was removed from the supplied plasmid by restriction endonuclease digestion with BspLU11I/PstI, gel-purified, and ligated into pTrcHis2B that had been digested with NcoI/PstI. The construct is cloned such that the stop codon in the insert is before the PstI site, which results in a construct in which the His-Tag is not attached to the isoprene synthase protein. The resulting plasmid pTrcPoplar (FIGS. 32 and 33; SEQ ID NO:15), was verified by sequencing.

Example 2B Demonstration of Isoprene Synthase Activity from Several Populus Isoprene Synthases

The following isoprene synthases were examined; Populus alba (Accession number BAD98243; FIGS. 137A and B; SEQ ID NO:30), Populus nigra (Accession number CAL69918; FIGS. 137C and D; SEQ ID NO:31), Populus tremuloides (Accession number AAQ16588; FIGS. 137 E, F, and G; SEQ ID NOs:32-33), Populus trichocarpa (Accession number ACD70404; FIGS. 137H and I; SEQ ID NO:34), Populus alba x Populus tremula (Accession number CAJ29303; FIGS. 137J and K; SEQ ID NO:35), and MCM112-Kudzu.

pET24Kudzu (also referred to as MCM112) was constructed as follows: the kudzu isoprene synthase gene was subcloned into the pET24d vector (Novagen) from the pCR2.1 vector (Invitrogen). The kudzu IspS gene was amplified from pTrcKudzu template DNA using primers MCM50 5′-GATCATGCAT TCGCCCTTAG GAGGTAAAAAAACATGTGTGCGACCTCTTC TCAATTTACT (SEQ ID NO:52); and MCM53 5′-CGGTCGACGGATCCCTGCAG TTAGACATAC ATCAGCTG (SEQ ID NO:50). PCR reactions were carried out using Taq DNA Polymerase (Invitrogen), and the resulting PCR product was cloned into pCR2.1-TOPO TA cloning vector (Invitrogen), and transformed into E. coli Top10 chemically competent cells (Invitrogen). Transformants were plated on L-agar containing carbenicillin (50 μg/ml) and incubated overnight at 37° C. Five ml Luria Broth cultures containing carbenicillin 50 μg/ml were inoculated with single transformants and grown overnight at 37° C. Five colonies were screened for the correct insert by sequencing of plasmid DNA isolated from 1 ml of liquid culture (Luria Broth) and purified using the QIAprep Spin Mini-prep Kit (Qiagen). The resulting plasmid, designated MCM93, contains the kudzu IspS coding sequence in a pCR2.1 backbone (FIG. 137L). The sequence of MCM93 (SEQ ID NO:36) is shown in FIGS. 137M and N.

The kudzu coding sequence was removed by restriction endonuclease digestion with PciI and BamH1 (Roche) and gel purified using the QIAquick Gel Extraction kit (Qiagen). The pET24d vector DNA was digested with NcoI and BamHI (Roche), treated with shrimp alkaline phosphatase (Roche), and purified using the QIAprep Spin Mini-prep Kit (Qiagen). The kudzu IspS fragment was ligated to the NcoI/BamH1 digested pET24d using the Rapid DNA Ligation Kit (Roche) at a 5:1 fragment to vector ratio in a total volume of 20 μl. A portion of the ligation mixture (5 μl) was transformed into E. coli Top 10 chemically competent cells and plated on L agar containing kanamycin (50 μg/ml). The correct transformant was confirmed by sequencing and transformed into chemically competent BL21(λDE3)pLysS cells (Novagen). A single colony was selected after overnight growth at 37° C. on L agar containing kanamycin (50 μg/ml). A map of the resulting plasmid designated as pET24D-Kudzu is shown in FIG. 137O. The sequence of pET24D-Kudzu (SEQ ID NO:37) is shown in FIGS. 137P and Q.

Escherichia coli optimized isoprene synthase genes cloned into the pET24a expression vector (Novagen) were purchased from DNA2.0 (Menlo Park, Calif.) for Populus tremuloides, Populus alba, Populus nigra and Populus trichocarpa. Genes were synthesized with the chloroplast transit peptide sequence removed, resulting in expression of mature proteins.

The construct for the Kudzu isoprene synthase was used as control in this example. The plasmids were transformed into the E. coli expression host BL21(DE3)plysS and transformants were grown in 0.6 ml TM3 medium. The recipe for TM3 medium is as follows: K₂HPO₄ (13.6 g/l) KH₂PO₄ (13.6 g/l), MgSO₄*7H₂O (2 g/L) Citric Acid Monohydrate (2 g/L) Ferric Ammonium Citrate (0.3 g/L) (NH₄)₂SO₄ (3.2 g/L) yeast extract (0.2 g/L) 1 ml of 1000× Trace Elements solution, pH adjusted to 6.8 with ammonium hydroxide qs to volume with sterile DI H₂O and filter sterilized with a 0.22 micron filter. The recipe for 1000× Trace Elements solution is as follows: Citric Acids*H₂O (40 g/L), MnSO₄*H₂O (30 g/L), NaCl (10 g/L), FeSO₄*7H₂O (1 g/L), CoCl₂*6H₂O (1 g/L), ZnSO₄*7H₂O (1 g/L), CuSO₄*5H₂O (100 mg/L), H₃BO₃ (100 mg/L), NaMoO₄*2H₂O (100 mg/L). Each component was dissolved one at a time in DI H₂O, pH adjusted to 3.0 with HCl/NaOH, qs to volume and filter sterilized with a 0.22 micron filter.

The cultures were induced with 400 μM IPTG and growth was continued to OD₆₀₀ of about 5. Aliquots of culture were transferred to a deep well glass plate and wells were sealed with aluminum plate sealer. The plate was incubated at 25° C. for 30 minutes with shaking at 450 rpm. The reactions were heat inactivated by raising the temperature to 70° C. for 5 minutes. Whole cell head space was measured by the GCMS method as described in Example 1, Part II.

K_(m) values were obtained from cultures grown in similar manner but cells were harvested and lysed by a freeze/thaw lysozyme protocol. A volume of 400 μl, of culture was transferred into a new 96-well plate (Perkin Elmer, Catalog No. 6008290) and cells were harvested by centrifugation in a Beckman Coulter Allegra 6R centrifuge at 2500×g. The pellet was resuspended in 200 mL of hypotonic buffer (5 mM MgCL₂, 5 mM Tris HCl, 5 mM DTT pH 8.0) and the plate was frozen at −80° C. for a minimum time of 60 minutes. Cell lysate was prepared by thawing the plate and adding 32 mL of isoprene synthase DMAPP assay buffer (57 mM Tris HCl, 19 mM MgCl₂, 74 mg/mL DNase I (Sigma Catalog No. DN-25), 2.63×10⁵ U/mL of ReadyLyse lysozyme solution (Epicentre Catalog No. R1802M), and 5 mg/mL of molecular biology grade BSA. The plate was incubated with shaking at 25° C. for 30 minutes and then placed on ice. DMAPP and lysate were added at desired concentration in a sealed deep well glass block for the whole cell head space assay described above. The reactions were allowed to proceed for 1 hour and then terminated by the heat step described above and head space activity was measured also as described.

In an alternate approach, the activity of the enzymes was measured from cells cultured in 25 mL volume and induced similarly as described above. Cells were harvested by centrifugation and the pellets were lysed by French pressing in buffer consisting of 50% glycerol mixed 1:1 with 20 mM Tris/HCl pH 7.4, 20 mM MgCl₂, 200 mM KCl, 1 mM DTT. A lysate volume of 25 μL was assayed for isoprene synthase activity in 2 mL screw cap vials containing 75 μL of assay buffer (66.6 mM Tris/HCl pH 8, 6.66 mM DMAPP, 43 mM, MgCl₂). The reaction was incubated for 15 minutes at 30° C. and was quenched by the addition of 100 μL of 250 mM EDTA through the septum of the vial. Isoprene was measured by GC/MS as described in Example 1, Part II.

All methods for the determination of activity showed that the poplar enzyme derived from the pure bred poplars were several-fold higher than the Populus [alba x tremula]. FIGS. 138 and 139 showed these results for the whole cell head space assay and the DMAPP assay, respectively, and surprisingly indicate that enzymes from P. nigra, P. tremuloides, P. trichocarpa, and P. alba all had significantly higher activity than hybrid [P. alba x P. tremula].

The DMAPP assay was performed as follows: a volume of 400 μL of culture was transferred into a new 96-well plate (Perkin Elmer, Catalog No. 6008290) and cells were harvested by centrifugation in a Beckman Coulter Allegra 6R centrifuge at 2500×g. The pellet was resuspended in 200 mL of hypotonic buffer (5 mM MgCL₂, 5 mM Tris HCl, 5 mM DTT pH 8.0) and the plate was frozen at −80° C. for a minimum time of 60 minutes. Cell lysate was prepared by thawing the plate and adding 32 mL of isoprene synthase DMAPP assay buffer (57 mM Tris HCl, 19 mM MgCl₂, 74 mg/mL DNase I (Sigma Catalog No. DN-25), 2.63×10⁵ U/mL of ReadyLyse lysozyme solution (Epicentre Catalog No. R1802M), and 5 mg/mL of molecular biology grade BSA. The plate was incubated with shaking at 25° C. for 30 minutes and then placed on ice. For isoprene production an 80 mL aliquot of lysate was transferred to a 96-deep well glass plate (Zinsser Catalog No. 3600600) and 20 mL of a 10 mM DMAPP solution in 100 mM KHPO₄, pH 8.2 (Cayman Chemical Catalog No. 63180) was added. The plate was sealed with an aluminum plate seal (Beckman Coultor Catalog No. 538619) and incubated with shaking at 30° C. for 60 minutes. The enzymatic reactions were terminated by heating the glass block (70° C. for 5 minutes). The cell head space of each well was quantitatively analyzed as described in Example 1, Part II.

Notably, P. alba, P. tremuloides, P. trichocarpa had higher activity than the isoprene synthase from Kudzu. The enzyme from P. alba was expressed with the greatest activity of all enzymes tested. The higher activities observed with the cell lysate compared to the whole cell head space assay was likely due to limitations in DMAPP, the substrate for these enzymes, delivered by the endogenous deoxyxylulose 5-phosphate (DXP) pathway of the cell.

K_(m) kinetic parameter was measured to be about 2 to 3 mM for all enzymes for which the value was determined.

Example 3 Production of Isoprene in Panteoa citrea Expressing Recombinant Kudzu Isoprene Synthase

The pTrcKudzu and pCL-lac Kudzu plasmids described in Example 1 were electroporated into P. citrea (U.S. Pat. No. 7,241,587). Transformants were selected on LA containing carbenicillin (200 μg/ml) or spectinomycin (50 μg/ml) respectively. Production of isoprene from shake flasks and determination of the amount of isoprene produced was performed as described in Example 1 for E. coli strains expressing recombinant kudzu isoprene synthase. Results are shown in FIG. 10.

Example 4 Production of Isoprene in Bacillus subtilis Expressing Recombinant Kudzu Isoprene Synthase

I. Construction of a B. subtilis Replicating Plasmid for the Expression of Kudzu Isoprene Synthase

The kudzu isoprene synthase gene was expressed in Bacillus subtilis aprEnprE Pxyl-comK strain (BG3594comK) using a replicating plasmid (0519 with a chloramphenicol resistance cassette) under control of the aprE promoter. The isoprene synthase gene, the aprE promoter and the transcription terminator were amplified separately and fused using PCR. The construct was then cloned into pBS19 and transformed into B. subtilis.

a) Amplification of the aprE Promoter

The aprE promoter was amplified from chromosomal DNA from Bacillus subtilis using the following primers:

CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 53) 5′-GACATCAATTGCTCCATTTTCTTCTGCTATC CF 07-43 (−) Fuse aprE promoter to Kudzu ispS (SEQ ID NO: 54) 5′-ATTGAGAAGAGGTCGCACACACTCTTTACCCTCTCCTTTTA b) Amplification of the Isoprene Synthase Gene

The kudzu isoprene synthase gene was amplified from plasmid pTrcKudzu (SEQ ID NO:2). The gene had been codon optimized for E. coli and synthesized by DNA 2.0. The following primers were used:

CF 07-42 (+) Fuse the aprE promoter to kudzu isoprene synthase gene (GTG start codon) (SEQ ID NO: 55) 5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT CF 07-45 (−) Fuse the 3′ end of kudzu isoprene synthase gene to the terminator (SEQ ID NO: 56) 5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATC c) Amplification of the Transcription Terminator

The terminator from the alkaline serine protease of Bacillus amyliquefaciens was amplified from a previously sequenced plasmid pJHPms382 using the following primers:

CF 07-44 (+) Fuse the 3′ end of kudzu isoprene synthase to the terminator (SEQ ID NO: 57) 5′-GATTAACCAGCTGATGTATGTCTAAAAAAAACCGGCCTTGG CF 07-46 (−) End of B. amyliquefaciens terminator (BamHI) (SEQ ID NO: 58) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The kudzu fragment was fused to the terminator fragment using PCR with the following primers:

CF 07-42 (+) Fuse the aprE promoter to kudzu isoprene synthase gene (GTG start codon) (SEQ ID NO: 55) 5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT CF 07-46 (−) End of B. amyliquefaciens terminator (BamHI) 5′- GACATGACGGATCCGATTACGAATGCCGTCTC (SEQ ID NO: 58)

The kudzu-terminator fragment was fused to the promoter fragment using PCR with the following primers:

CF 797 (+) Start aprE promoter MfeI 5′- GACATCAATTGCTCCATTTTCTTCTGCTATC (SEQ ID NO: 53) CF 07-46 (−) End of B. amyliquefaciens terminator (BamHI) 5′- GACATGACGGATCCGATTACGAATGCCGTCTC (SEQ ID NO: 58)

The fusion PCR fragment was purified using a Qiagen kit and digested with the restriction enzymes MfeI and BamHI. This digested DNA fragment was gel purified using a Qiagen kit and ligated to a vector known as pBS19, which had been digested with EcoRI and BamHI and gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonies were selected on LA+50 carbenicillin plates. A total of six colonies were chosen and grown overnight in LB+50 carbenicillin and then plasmids were isolated using a Qiagen kit. The plasmids were digested with EcoRI and BamHI to check for inserts and three of the correct plasmids were sent in for sequencing with the following primers:

CF 149 (+) EcoRI start of aprE promoter 5′-GACATGAATTCCTCCATTTTCTTCTGC (SEQ ID NO: 59) CF 847 (+) Sequence in pXX 049 (end of aprE promoter) 5′-AGGAGAGGGTAAAGAGTGAG (SEQ ID NO: 60) CF 07-45 (−) Fuse the 3′ end of kudzu isoprene synthase to the terminator (SEQ ID NO: 56) 5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATC CF 07-48 (+) Sequencing primer for kudzu isoprene synthase 5′-CTTTTCCATCACCCACCTGAAG (SEQ ID NO: 61) CF 07-49 (+) Sequencing in kudzu isoprene synthase 5′-GGCGAAATGGTCCAACAACAAAATTATC (SEQ ID NO: 62)

The plasmid designated pBS Kudzu #2 (FIGS. 52 and 12; SEQ ID NO:5) was correct by sequencing and was transformed into BG 3594 comK, a Bacillus subtilis host strain. Selection was done on LA+5 chloramphenicol plates. A transformant was chosen and struck to single colonies on LA+5 chloramphenicol, then grown in LB+5 chloramphenicol until it reached an OD₆₀₀ of 1.5. It was stored frozen in a vial at −80° C. in the presence of glycerol. The resulting strain was designated CF 443.

II. Production of Isoprene in Shake Flasks Containing B. subtilis Cells Expressing Recombinant Isoprene Synthase

Overnight cultures were inoculated with a single colony of CF 443 from a LA+Chloramphenicol (Cm, 25 μg/ml). Cultures were grown in LB+Cm at 37° C. with shaking at 200 rpm. These overnight cultures (1 ml) were used to inoculate 250 ml baffled shake flasks containing 25 ml Grants II media and chloramphenicol at a final concentration of 25 μg/ml. Grants II Media recipe was 10 g soytone, 3 ml 1M K₂HPO₄, 75 g glucose, 3.6 g urea, 100 ml 10×MOPS, q.s. to 1 L with H₂O, pH 7.2; 10×MOPS recipe was 83.72 g MOPS, 7.17 g tricine, 12 g KOH pellets, 10 ml 0.276M K₂SO₄ solution, 10 ml 0.528M MgCl₂ solution, 29.22 g NaCl, 100 ml 100× micronutrients, q.s. to 1 L with H₂O; and 100× micronutrients recipe was 1.47 g CaCl₂*2H₂O, 0.4 g FeSO₄.7H₂O, 0.1 g MnSO₄*H₂O, 0.1 g ZnSO₄*H₂O, 0.05 g CuCl₂*2H₂O, 0.1 g CoCl₂*6H₂O, 0.1 g Na₂MoO₄.2H₂O, q.s. to 1 L with H₂O, Shake flasks were incubated at 37° C. and samples were taken at 18, 24, and 44 hours. At 18 hours the headspaces of CF443 and the control strain were sampled. This represented 18 hours of accumulation of isoprene. The amount of isoprene was determined by gas chromatography as described in Example 1. Production of isoprene was enhanced significantly by expressing recombinant isoprene synthase (FIG. 11).

III. Production of Isoprene by CF443 in 14 L Fermentation

Large scale production of isoprene from B. subtilis containing the recombinant kudzu isoprene synthase gene on a replication plasmid was determined from a fed-batch culture. Bacillus strain CF 443, expressing a kudzu isoprene synthase gene, or control stain which does not express a kudzu isoprene synthase gene were cultivated by conventional fed-batch fermentation in a nutrient medium containing soy meal (Cargill), sodium and potassium phosphate, magnesium sulfate and a solution of citric acid, ferric chloride and manganese chloride. Prior to fermentation the media is macerated for 90 minutes using a mixture of enzymes including cellulases, hemicellulases and pectinases (see, WO95/04134). 14-L batch fermentations are fed with 60% wt/wt glucose (Cargill DE99 dextrose, ADM Versadex greens or Danisco invert sugar) and 99% wt/wt oil (Western Family soy oil, where the 99% wt/wt is the concentration of oil before it was added to the cell culture medium). Feed was started when glucose in the batch was non-detectable. The feed rate was ramped over several hours and was adjusted to add oil on an equal carbon basis. The pH was controlled at 6.8-7.4 using 28% w/v ammonium hydroxide. In case of foaming, antifoam agent was added to the media. The fermentation temperature was controlled at 37° C. and the fermentation culture was agitated at 750 rpm. Various other parameters such as pH, D0%, airflow, and pressure were monitored throughout the entire process. The DO % is maintained above 20. Samples were taken over the time course of 36 hours and analyzed for cell growth (OD₅₅₀) and isoprene production. Results of these experiments are presented in FIGS. 53A and 53B.

IV. Integration of the Kudzu Isoprene Synthase (ispS) in B. subtilis.

The kudzu isoprene synthase gene was cloned in an integrating plasmid (pJH101-cmpR) under the control of the aprE promoter. Under the conditions tested, no isoprene was detected.

Example 5 Production of Isoprene in Trichoderma

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthase in Trichoderma reesei

The Yarrowia lipolytica codon-optimized kudzu IS gene was synthesized by DNA 2.0 (SEQ ID NO:6) (FIG. 13). This plasmid served as the template for the following PCR amplification reaction: 1 μl plasmid template (20 ng/ul), 1 μl Primer EL-945 (10 μM) 5′-GCTTATGGATCCTCTAGACTATTACACGTACATCAATTGG (SEQ ID NO:63), 1 μl Primer EL-965 (10 μM) 5′-CACCATGTGTGCAACCTCCTCCCAGTTTAC (SEQ ID NO:64), 1 μl dNTP (10 mM), 5 μl 10×PfuUltra II Fusion HS DNA Polymerase Buffer, 1 μl PfuUltra II Fusion HS DNA Polymerase, 40 μl water in a total reaction volume of 50 μl. The forward primer contained an additional 4 nucleotides at the 5′-end that did not correspond to the Y. lipolytica codon-optimized kudzu isoprene synthase gene, but was required for cloning into the pENTR/D-TOPO vector. The reverse primer contained an additional 21 nucleotides at the 5′-end that did not correspond to the Y. lipolytica codon-optimized kudzu isoprene synthase gene, but were inserted for cloning into other vector backbones. Using the MJ Research PTC-200 Thermocycler, the PCR reaction was performed as follows: 95° C. for 2 minutes (first cycle only), 95° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 30 seconds (repeat for 27 cycles), 72° C. for 1 minute after the last cycle. The PCR product was analyzed on a 1.2% E-gel to confirm successful amplification of the Y. lipolytica codon-optimized kudzu isoprene synthase gene.

The PCR product was then cloned using the TOPO pENTR/D-TOPO Cloning Kit following manufacturer's protocol: 1 μl PCR reaction, 1 μl Salt solution, 1 μl TOPO pENTR/D-TOPO vector and 3 μl water in a total reaction volume of 6 μl. The reaction was incubated at room temperature for 5 minutes. One microliter of TOPO reaction was transformed into TOP10 chemically competent E. coli cells. The transformants were selected on LA+50 μg/ml kanamycin plates. Several colonies were picked and each was inoculated into a 5 ml tube containing LB+50 μg/ml kanamycin and the cultures grown overnight at 37° C. with shaking at 200 rpm. Plasmids were isolated from the overnight culture tubes using QIAprep Spin Miniprep Kit, following manufacturer's protocol. Several plasmids were sequenced to verify that the DNA sequence was correct.

A single pENTR/D-TOPO plasmid, encoding a Y. lipolytica codon-optimized kudzu isoprene synthase gene, was used for Gateway Cloning into a custom-made pTrex3g vector. Construction of pTrex3g is described in WO 2005/001036 A2. The reaction was performed following manufacturer's protocol for the Gateway LR Clonase II Enzyme Mix Kit (Invitrogen): 1 μl Y. lipolytica codon-optimized kudzu isoprene synthase gene pENTR/D-TOPO donor vector, 1 μl pTrex3g destination vector, 6 μl TE buffer, pH 8.0 in a total reaction volume of 8 μl. The reaction was incubated at room temperature for 1 hour and then 1 μl proteinase K solution was added and the incubation continued at 37° C. for 10 minutes. Then 1 μl of reaction was transformed into TOP10 chemically competent E. coli cells. The transformants were selected on LA+50 μg/ml carbenicillin plates. Several colonies were picked and each was inoculated into a 5 ml tube containing LB+50 μg/ml carbenicillin and the cultures were grown overnight at 37° C. with shaking at 200 rpm. Plasmids were isolated from the overnight culture tubes using QIAprep Spin Miniprep Kit (Qiagen, Inc.), following manufacturer's protocol. Several plasmids were sequenced to verify that the DNA sequence was correct.

Biolistic transformation of Y. lipolytica codon-optimized kudzu isoprene synthase pTrex3g plasmid (FIG. 14) into a quad delete Trichoderma reesei strain was performed using the Biolistic PDS-1000/HE Particle Delivery System (see WO 2005/001036 A2). Isolation of stable transformants and shake flask evaluation was performed using protocol listed in Example 11 of patent publication WO 2005/001036 A2.

II. Production of Isoprene in Recombinant Strains of T. reesei

One ml of 15 and 36 hour old cultures of isoprene synthase transformants described above were transferred to head space vials. The vials were sealed and incubated for 5 hours at 30° C. Head space gas was measured and isoprene was identified by the method described in Example 1. Two of the transformants showed traces of isoprene. The amount of isoprene could be increased by a 14 hour incubation. The two positive samples showed isoprene at levels of about 0.5 μg/L for the 14 hour incubation. The untransformed control showed no detectable levels of isoprene. This experiment shows that T. reesei is capable of producing isoprene from endogenous precursor when supplied with an exogenous isoprene synthase.

Example 6 Production of Isoprene in Yarrowia

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthase in Yarrowia lipolytica.

The starting point for the construction of vectors for the expression of the kudzu isoprene synthase gene in Yarrowia lipolytica was the vector pSPZ1(MAP29Spb). The complete sequence of this vector (SEQ ID NO:7) is shown in FIG. 15.

The following fragments were amplified by PCR using chromosomal DNA of a Y. lipolytica strain GICC 120285 as the template: a promotorless form of the URA3 gene, a fragment of 18S ribosomal RNA gene, a transcription terminator of the Y. lipolytica XPR2 gene and two DNA fragments containing the promoters of XPR2 and ICL1 genes. The following PCR primers were used:

ICL1 3 (SEQ ID NO: 65) 5′-GGTGAATTCAGTCTACTGGGGATTCCCAAATCTATATATACTGCAGG TGAC ICL1 5 5′-GCAGGTGGGAAACTATGCACTCC (SEQ ID NO: 66) XPR 3 5′- CCTGAATTCTGTTGGATTGGAGGATTGGATAGTGGG (SEQ ID NO: 67) XPR 5 5′-GGTGTCGACGTACGGTCGAGCTTATTGACC (SEQ ID NO: 68) XPRT3 5′-GGTGGGCCCGCATTTTGCCACCTACAAGCCAG (SEQ ID NO: 69) XPRT 5 (SEQ ID NO: 70) 5′-GGTGAATTCTAGAGGATCCCAACGCTGTTGCCTACAACGG Y18S3 5′- GGTGCGGCCGCTGTCTGGACCTGGTGAGTTTCCCCG (SEQ ID NO: 71) Y18S 5 5′- GGTGGGCCCATTAAATCAGTTATCGTTTATTTGATAG (SEQ ID NO: 72) YURA3 5′- GGTGACCAGCAAGTCCATGGGTGGTTTGATCATGG (SEQ ID NO: 73) YURA 50 5′- GGTGCGGCCGCCTTTGGAGTACGACTCCAACTATG (SEQ ID NO: 74) YURA 51 5′-GCGGCCGCAGACTAAATTTATTTCAGTCTCC (SEQ ID NO: 75)

For PCR amplification the PfuUltraII polymerase (Stratagene), supplier-provided buffer and dNTPs, 2.5 μM primers and the indicated template DNA were used as per the manufacturer's instructions. The amplification was done using the following cycle: 95° C. for 1 min; 34× (95° C. for 30 sec; 55° C. for 30 sec; 72° C. for 3 min) and 10 min at 72° C. followed by a 4° C. incubation.

Synthetic DNA molecules encoding the kudzu isoprene synthase gene, codon-optimized for expression in Yarrowia, was obtained from DNA 2.0 (FIG. 16; SEQ ID NO:8). Full detail of the construction scheme of the plasmids pYLA(KZ1) and pYLI(KZ1) carrying the synthetic kudzu isoprene synthase gene under control of XPR2 and ICL1 promoters respectively is presented in FIG. 18. Control plasmids in which a mating factor gene (MAP29) is inserted in place of an isoprene synthase gene were also constructed (FIGS. 18E and 18F).

A similar cloning procedure can be used to express a poplar (Populus alba x Populus tremula) isoprene synthase gene. The sequence of the poplar isoprene is described in Miller B. et al. (2001) Planta 213, 483-487 and shown in FIG. 17 (SEQ ID NO:9). A construction scheme for the generation the plasmids pYLA(POP1) and pYLI(POP1) carrying synthetic poplar isoprene synthase gene under control of XPR2 and ICL1 promoters respectively is presented in FIGS. 18A and B.

II. Production of Isoprene by Recombinant Strains of Y. lipolytica.

Vectors pYLA(KZ1), pYLI(KZ1), pYLA(MAP29) and pYLI(MAP29) were digested with SacII and used to transform the strain Y. lipolytica CLIB 122 by a standard lithium acetate/polyethylene glycol procedure to uridine prototrophy. Briefly, the yeast cells grown in YEPD (1% yeast extract, 2% peptone, 2% glucose) overnight, were collected by centrifugation (4000 rpm, 10 min), washed once with sterile water and suspended in 0.1 M lithium acetate, pH 6.0. Two hundred μl aliquots of the cell suspension were mixed with linearized plasmid DNA solution (10-20 μg), incubated for 10 minutes at room temperature and mixed with 1 ml of 50% PEG 4000 in the same buffer. The suspensions were further incubated for 1 hour at room temperature followed by a 2 minutes heat shock at 42° C. Cells were then plated on SC his leu plates (0.67% yeast nitrogen base, 2% glucose, 100 mg/L each of leucine and histidine). Transformants appeared after 3-4 days of incubation at 30° C.

Three isolates from the pYLA(KZ1) transformation, three isolates from the pYLI(KZ1) transformation, two isolates from the pYLA(MAP29) transformation and two isolates from the pYLI(MAP29) transformation were grown for 24 hours in YEP7 medium (1% yeast extract, 2% peptone, pH 7.0) at 30° C. with shaking Cells from 10 ml of culture were collected by centrifugation, resuspended in 3 ml of fresh YEP7 and placed into 15 ml screw cap vials. The vials were incubated overnight at room temperature with gentle (60 rpm) shaking. Isoprene content in the headspace of these vials was analyzed by gas chromatography using mass-spectrometric detector as described in Example 1. All transformants obtained with pYLA(KZ1) and pYLI(KZ1) produced readily detectable amounts of isoprene (0.5 μg/L to 1 μg/L, FIG. 20). No isoprene was detected in the headspace of the control strains carrying phytase gene instead of an isoprene synthase gene.

Example 7 Production of Isoprene in E. coli Expressing Kudzu Isoprene Synthase and idi, or dxs, or idi and dxs

I. Construction of Vectors Encoding Kudzu Isoprene Synthase and idi, or dxs, or idi and dxs for the Production of Isoprene in E. coli

i) Construction of pTrcKudzuKan

The bla gene of pTrcKudzu (described in Example 1) was replaced with the gene conferring kanamycin resistance. To remove the bla gene, pTrcKudzu was digested with BspHI, treated with Shrimp Alkaline Phosphatase (SAP), heat killed at 65° C., then end-filled with Klenow fragment and dNTPs. The 5 kbp large fragment was purified from an agarose gel and ligated to the kan^(r) gene which had been PCR amplified from pCR-Blunt-II-TOPO using primers MCM22 5′-GATCAAGCTTAACCGGAATTGCCAGCTG (SEQ ID NO:76) and MCM23 5′-GATCCGATCGTCAGAAGAACTCGTCAAGAAGGC (SEQ ID NO:77), digested with HindIII and PvuI, and end-filled. A transformant carrying a plasmid conferring kanamycin resistance (pTrcKudzuKan) was selected on LA containing kanamycin 50 μg/ml.

ii) Construction of pTrcKudzu yIDI Kan

pTrcKudzuKan was digested with PstI, treated with SAP, heat killed and gel purified. It was ligated to a PCR product encoding idi from S. cerevisiae with a synthetic RBS. The primers for PCR were NsiI-YIDI 1 F 5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC (SEQ ID NO:78) and PstI-YIDI 1 R 5′-CCTTCTGCAGGACGCGTTGTTATAGC (SEQ ID NO:79); and the template was S. cerevisiae genomic DNA. The PCR product was digested with NsiI and PstI and gel purified prior to ligation. The ligation mixture was transformed into chemically competent TOP10 cells and selected on LA containing 50 μg/ml kanamycin. Several transformants were isolated and sequenced and the resulting plasmid was called pTrcKudzu-yIDI(kan) (FIGS. 34 and 35; SEQ ID NO:16).

iii) Construction of pTrcKudzu DXS Kan

Plasmid pTrcKudzuKan was digested with PstI, treated with SAP, heat killed and gel purified. It was ligated to a PCR product encoding dxs from E. coli with a synthetic RBS. The primers for PCR were MCM13 5′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCC G (SEQ ID NO:80) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:81); and the template was E. coli genomic DNA. The PCR product was digested with NsiI and PstI and gel purified prior to ligation. The resulting transformation reaction was transformed into TOP10 cells and selected on LA with kanamycin 50 μg/ml. Several transformants were isolated and sequenced and the resulting plasmid was called pTrcKudzu-DXS(kan) (FIGS. 36 and 37; SEQ ID NO:17).

iv) Construction of pTrcKudzu-yIDI-dxs (kan)

pTrcKudzu-yIDI(kan) was digested with PstI, treated with SAP, heat killed and gel purified. It was ligated to a PCR product encoding E. coli dxs with a synthetic RBS (primers MCM13 5′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCC G (SEQ ID NO:80) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:81); template TOP10 cells) which had been digested with NsiI and PstI and gel purified. The final plasmid was called pTrcKudzu-yIDI-dxs (kan) (FIGS. 21 and 22; SEQ ID NO:10).

v) Construction of pCL PtrcKudzu

A fragment of DNA containing the promoter, structural gene and terminator from Example 1 above was digested from pTrcKudzu using SspI and gel purified. It was ligated to pCL1920 which had been digested with PvuII, treated with SAP and heat killed. The resulting ligation mixture was transformed into TOP10 cells and selected in LA containing spectinomycin 50 μg/ml. Several clones were isolated and sequenced and two were selected. pCL PtrcKudzu and pCL PtrcKudzu (A3) have the insert in opposite orientations (FIGS. 38-41; SEQ ID NOs:18-19).

vi) Construction of pCL PtrcKudzu yIDI

The NsiI-PstI digested, gel purified, IDI PCR amplicon from (ii) above was ligated into pCL PtrcKudzu which had been digested with PstI, treated with SAP, and heat killed. The ligation mixture was transformed into TOP10 cells and selected in LA containing spectinomycin 50 μg/ml. Several clones were isolated and sequenced and the resulting plasmid is called pCL PtrcKudzu yIDI (FIGS. 42 and 43; SEQ ID NO:20).

vii) Construction of pCL PtrcKudzu DXS

The NsiI-PstI digested, gel purified, DXS PCR amplicon from (iii) above was ligated into pCL PtrcKudzu (A3) which had been digested with PstI, treated with SAP, and heat killed. The ligation mixture was transformed into TOP10 cells and selected in LA containing spectinomycin 50 μg/ml. Several clones were isolated and sequenced and the resulting plasmid is called pCL PtrcKudzu DXS (FIGS. 44 and 45; SEQ ID NO:21).

II. Measurement of Isoprene in Headspace from Cultures Expressing Kudzu Isoprene Synthase, idi, and/or dxs at Different Copy Numbers.

Cultures of E. coli BL21(λDE3) previously transformed with plasmids pTrcKudzu(kan) (A), pTrcKudzu-yIDI kan (B), pTrcKudzu-DXS kan (C), pTrcKudzu-yIDI-DXS kan (D) were grown in LB kanamycin 50 μg/mL. Cultures of pCL PtrcKudzu (E), pCL PtrcKudzu, pCL PtrcKudzu-yIDI (F) and pCL PtrcKudzu-DXS (G) were grown in LB spectinomycin 50 μg/mL. Cultures were induced with 400 μM IPTG at time 0 (OD₆₀₀ approximately 0.5) and samples taken for isoprene headspace measurement (see Example 1). Results are shown in FIG. 23A-23G.

Plasmid pTrcKudzu-yIDI-dxs (kan) was introduced into E. coli strain BL21 by transformation. The resulting strain BL21/pTrc Kudzu IDI DXS was grown overnight in LB containing kanamycin (50 μg/ml) at 20° C. and used to inoculate shake flasks of TM3 (13.6 g K₂PO₄, 13.6 g KH₂PO₄, 2.0 g MgSO₄.7H₂O), 2.0 g citric acid monohydrate, 0.3 g ferric ammonium citrate, 3.2 g (NH₄)₂SO₄, 0.2 g yeast extract, 1.0 ml 1000× Modified Trace Metal Solution, adjusted to pH 6.8 and q.s. to H₂O, and filter sterilized) containing 1% glucose. Flasks were incubated at 30° C. until an OD₆₀₀ of 0.8 was reached, and then induced with 400 μM IPTG. Samples were taken at various times after induction and the amount of isoprene in the head space was measured as described in Example 1. Results are shown in FIG. 23H.

III. Production of Isoprene from Biomass in E. coli/pTrcKudzu yIDI DXS

The strain BL21 pTrcKudzuIDIDXS was tested for the ability to generate isoprene from three types of biomass; bagasse, corn stover and soft wood pulp with glucose as a control. Hydrolysates of the biomass were prepared by enzymatic hydrolysis (Brown, L. and Torget, R., 1996, NREL standard assay method Lap-009 “Enzymatic Saccharification of Lignocellulosic Biomass”) and used at a dilution based upon glucose equivalents. In this example, glucose equivalents were equal to 1% glucose. A single colony from a plate freshly transformed cells of BL21 (DE3) pTrcKudzu yIDI DXS (kan) was used to inoculate 5 ml of LB plus kanamycin (50 μg/ml). The culture was incubated overnight at 25° C. with shaking. The following day the overnight culture was diluted to an OD₆₀₀ of 0.05 in 25 ml of TM3+0.2% YE+1% feedstock. The feedstock was corn stover, bagasse, or softwood pulp. Glucose was used as a positive control and no glucose was used as a negative control. Cultures were incubated at 30° C. with shaking at 180 rpm. The culture was monitored for OD₆₀₀ and when it reached an OD₆₀₀ of ˜0.8, cultures were analyzed at 1 and 3 hours for isoprene production as described in Example 1. Cultures are not induced. All cultures containing added feedstock produce isoprene equivalent to those of the glucose positive control. Experiments were done in duplicate and are shown in FIG. 46.

IV. Production of Isoprene from Invert Sugar in E. coli/pTrcKudzuIDIDXS

A single colony from a plate freshly transformed cells of BL21 (ADE3)/pTrcKudzu yIDI DXS (kan) was used to inoculate 5 mL of LB+kanamycin (50 μg/ml). The culture was incubated overnight at 25° C. with shaking. The following day the overnight culture was diluted to an OD₆₀₀ of 0.05 in 25 ml of TM3+0.2% YE+1% feedstock. Feedstock was glucose, inverted glucose or corn stover. The invert sugar feedstock (Danisco Invert Sugar) was prepared by enzymatically treating sucrose syrup. AFEX corn stover was prepared as described below (Part V). The cells were grown at 30° C. and the first sample was measured when the cultures reached an OD₆₀₀˜0.8-1.0 (0 hour). The cultures were analyzed for growth as measured by OD₆₀₀ and for isoprene production as in Example 1 at 0, 1 and 3 hours. Results are shown in FIG. 47.

V. Preparation of Hydrolysate from AFEX Pretreated Corn Stover

AFEX pretreated corn stover was obtained from Michigan Biotechnology Institute. The pretreatment conditions were 60% moisture, 1:1 ammonia loading, and 90° C. for 30 minutes, then air dried. The moisture content in the AFEX pretreated corn stover was 21.27%. The contents of glucan and xylan in the AFEX pretreated corn stover were 31.7% and 19.1% (dry basis), respectively. The saccharification process was as follows; 20 g of AFEX pretreated corn stover was added into a 500 ml flask with 5 ml of 1 M sodium citrate buffer pH 4.8, 2.25 ml of Accellerase 1000, 0.1 ml of Grindamyl H121 (Danisco xylanase product from Aspergillus niger for bread-making industry), and 72.65 ml of DI water. The flask was put in an orbital shaker and incubated at 50° C. for 96 hours. One sample was taken from the shaker and analyzed using HPLC. The hydrolysate contained 38.5 g/l of glucose, 21.8 g/l of xylose, and 10.3 g/l of oligomers of glucose and/or xylose.

VI. The Effect of Yeast Extract on Isoprene Production in E. coli Grown in Fed-Batch Culture

Fermentation was performed at the 14-L scale as previously described with E. coli cells containing the pTrcKudzu yIDI DXS plasmid described above. Yeast extract (Bio Springer, Montreal, Quebec, Canada) was fed at an exponential rate. The total amount of yeast extract delivered to the fermentor was varied between 70-830 g during the 40 hour fermentation. Optical density of the fermentation broth was measured at a wavelength of 550 nm. The final optical density within the fermentors was proportional to the amount of yeast extract added (FIG. 48A). The isoprene level in the off-gas from the fermentor was determined as previously described. The isoprene titer increased over the course of the fermentation (FIG. 48B). The amount of isoprene produced was linearly proportional to the amount of fed yeast extract (FIG. 48C).

VII. Production of Isoprene in 500 L Fermentation of pTrcKudzu DXS yIDI

A 500 liter fermentation of E. coli cells with a kudzu isoprene synthase, S. cerevisiae IDI, and E. coli DXS nucleic acids (E. coli BL21 (λDE3) pTrc Kudzu dxs yidi) was used to produce isoprene. The levels of isoprene varied from 50 to 300 μg/L over a time period of 15 hours. On the basis of the average isoprene concentrations, the average flow through the device and the extent of isoprene breakthrough, the amount of isoprene collected was calculated to be approximately 17 g.

VIII. Production of Isoprene in 500 L Fermentation of E. coli Grown in Fed-Batch Culture

Medium Recipe (Per Liter Fermentation Medium):

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium gas (NH₃) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotic were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in DI H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with 0.22 micron filter.

Fermentation was performed in a 500-L bioreactor with E. coli cells containing the pTrcKudzu yIDI DXS plasmid. This experiment was carried out to monitor isoprene formation from glucose and yeast extract at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain taken from a frozen vial was prepared in soytone-yeast extract-glucose medium. After the inoculum grew to OD 0.15, measured at 550 nm, 20 ml was used to inoculate a bioreactor containing 2.5-L soytone-yeast extract-glucose medium. The 2.5-L bioreactor was grown at 30° C. to OD 1.0 and 2.0-L was transferred to the 500-L bioreactor.

Yeast extract (Bio Springer, Montreal, Quebec, Canada) and glucose were fed at exponential rates. The total amount of glucose and yeast extract delivered to the bioreactor during the 50 hour fermentation was 181.2 kg and 17.6 kg, respectively. The optical density within the bioreactor over time is shown in FIG. 49A. The isoprene level in the off-gas from the bioreactor was determined as previously described. The isoprene titer increased over the course of the fermentation (FIG. 49B). The total amount of isoprene produced during the 50 hour fermentation was 55.1 g and the time course of production is shown in FIG. 49C.

Example 8 Production of Isoprene in E. coli Expressing Kudzu Isoprene Synthase and Recombinant Mevalonic Acid Pathway Genes

I. Cloning the Lower MVA Pathway

The strategy for cloning the lower mevalonic pathway was as follows. Four genes of the mevalonic acid biosynthesis pathway; mevalonate kinase (MVK), phosphomevalonate kinase (PMK), diphosphomevalonte decarboxylase (MVD) and isopentenyl diphosphate isomerase genes were amplified by PCR from S. cerevisiae chromosomal DNA and cloned individually into the pCR BluntII TOPO plasmid (Invitrogen). In some cases, the idi gene was amplified from E. coli chromosomal DNA. The primers were designed such that an E. coli consensus RBS (AGGAGGT (SEQ ID NO:82) or AAGGAGG (SEQ ID NO:83)) was inserted at the 5′ end, 8 bp upstream of the start codon and a PstI site was added at the 3′ end. The genes were then cloned one by one into the pTrcHis2B vector until the entire pathway was assembled.

Chromosomal DNA from S. cerevisiae S288C was obtained from ATCC (ATCC 204508D). The MVK gene was amplified from the chromosome of S. cerevisiae using primers MVKF (5′-AGGAGGTAAAAAAACATGTCATTACCGTTCTTAACTTCTGC, SEQ ID NO:84) and MVK-PstI-R (5′-ATGGCTGCAGGCCTATCGCAAATTAGCTTATGAAGTCCATGGTAAATTCGTG, SEQ ID NO:85) using PfuTurbo as per manufacturer's instructions. The correct sized PCR product (1370 bp) was identified by electrophoresis through a 1.2% E-gel (Invitrogen) and cloned into pZeroBLUNT TOPO. The resulting plasmid was designated pMVK1. The plasmid pMVK1 was digested with Sad and Taq1 restriction endonucleases and the fragment was gel purified and ligated into pTrcHis2B digested with Sad and BstBI. The resulting plasmid was named pTrcMVK1.

The second gene in the mevalonic acid biosynthesis pathway, PMK, was amplified by PCR using primers: PstI-PMK1 R (5′-GAATTCGCCCTTCTGCAGCTACC, SEQ ID NO:86) and BsiHKA I-PMK1 F (5′-CGACTGGTGCACCCTTAAGGAGGAAAAAAACATGTCAG, SEQ ID NO:87). The PCR reaction was performed using Pfu Turbo polymerase (Stratagene) as per manufacturer's instructions. The correct sized product (1387 bp) was digested with PstI and BsiHKI and ligated into pTrcMVK1 digested with PstI. The resulting plasmid was named pTrcKK. The MVD and the idi genes were cloned in the same manner. PCR was carried out using the primer pairs PstI-MVD 1 R (5′-GTGCTGGAATTCGCCCTTCTGCAGC, SEQ ID NO:88) and NsiI-MVD 1 F (5′-GTAGATGCATGCAGAATTCGCCCTTAAGGAGG, SEQ ID NO:89) to amplify the MVD gene and PstI-YIDI 1 R (5′-CCTTCTGCAGGACGCGTTGTTATAGC, SEQ ID NO:79) and NsiI-YIDI 1 F (5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC, SEQ ID NO:78) to amplify the yIDI gene. In some cases the IPP isomerase gene, idi from E. coli was used. To amplify idi from E. coli chromosomal DNA, the following primer set was used: PstI-CIDI 1 R (5′-GTGTGATGGATATCTGCAGAATTCG, SEQ ID NO:90) and NsiI-CIDI 1 F (5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAACATG, SEQ ID NO:91). Template DNA was chromosomal DNA isolated by standard methods from E. coli FM5 (WO 96/35796 and WO 2004/033646, which are each hereby incorporated by reference in their entireties, particularly with respect to isolation of nucleic acids). The final plasmids were named pKKDIy for the construct encoding the yeast idi gene or pKKDIc for the construct encoding the E. coli idi gene. The plasmids were transformed into E. coli hosts BL21 for subsequent analysis. In some cases the isoprene synthase from kudzu was cloned into pKKDIy yielding plasmid pKKDIyIS.

The lower MVA pathway was also cloned into pTrc containing a kanamycin antibiotic resistance marker. The plasmid pTrcKKDIy was digested with restriction endonucleases ApaI and PstI, the 5930 bp fragment was separated on a 1.2% agarose E-gel and purified using the Qiagen Gel Purification kit according to the manufacturer's instructions. The plasmid pTrcKudzuKan, described in Example 7, was digested with restriction endonucleases ApaI and PstI, and the 3338 bp fragment containing the vector was purified from a 1.2% E-gel using the Qiagen Gel Purification kit. The 3338 bp vector fragment and the 5930 bp lower MVA pathway fragment were ligated using the Roche Quick Ligation kit. The ligation mix was transformed into E. coli TOP10 cells and tranformants were grown at 37° C. overnight with selection on LA containing kanamycin (50 μg/ml). The transformants were verified by restriction enzyme digestion and one was frozen as a stock. The plasmid was designated pTrcKanKKDIy.

II. Cloning a Kudzu Isoprene Synthase Gene into pTrcKanKKDIy

The kudzu isoprene synthase gene was amplified by PCR from pTrcKudzu, described in Example 1, using primers MCM50 5′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGTGTGCGACCTCTTCTCAATTTAC T (SEQ ID NO:52) and MCM53 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:50). The resulting PCR fragment was cloned into pCR2.1 and transformed into E. coli TOP10. This fragment contains the coding sequence for kudzu isoprene synthase and an upstream region containing a RBS from E. coli. Transformants were incubated overnight at 37° C. with selection on LA containing carbenicillin (50 μg/ml). The correct insertion of the fragment was verified by sequencing and this strain was designated MCM93.

The plasmid from strain MCM93 was digested with restriction endonucleases NsiI and PstI to liberate a 1724 bp insert containing the RBS and kudzu isoprene synthase. The 1724 bp fragment was separated on a 1.2% agarose E-gel and purified using the Qiagen Gel Purification kit according to the manufacturer's instructions. Plasmid pTrcKanKKDIy was digested with the restriction endonuclease PstI, treated with SAP for 30 minutes at 37° C. and purified using the Qiagen PCR cleanup kit. The plasmid and kudzu isoprene synthase encoding DNA fragment were ligated using the Roche Quick Ligation kit. The ligation mix was transformed into E. coli TOP10 cells and transformants were grown overnight at 37° C. with selection on LA containing Kanamycin at 50 μg/ml. The correct transformant was verified by restriction digestion and the plasmid was designated pTrcKKDyIkISKan (FIGS. 24 and 25; SEQ ID NO:11). This plasmid was transformed into BL21(λDE3) cells (Invitrogen).

III. Isoprene Production from Mevalonate in E. coli Expressing the Recombinant Lower Mevalonate Pathway and Isoprene Synthase from Kudzu.

Strain BL21/pTrcKKDyIkISKan was cultured in MOPS medium (Neidhardt et al., (1974) J. Bacteriology 119:736-747) adjusted to pH 7.1 and supplemented with 0.5% glucose and 0.5% mevalonic acid. A control culture was also set up using identical conditions but without the addition of 0.5% mevalonic acid. The culture was started from an overnight seed culture with a 1% inoculum and induced with 500 μM IPTG when the culture had reached an OD₆₀₀ of 0.3 to 0.5. The cultures were grown at 30° C. with shaking at 250 rpm. The production of isoprene was analyzed 3 hours after induction by using the head space assay described in Example 1. Maximum production of isoprene was 6.67×10⁻⁴ mol/L_(broth)/OD₆₀₀/hr where L_(broth) is the volume of broth and includes both the volume of the cell medium and the volume of the cells. The control culture not supplemented with mevalonic acid did not produce measurable isoprene.

IV. Cloning the Upper MVA Pathway

The upper mevalonate biosynthetic pathway, comprising two genes encoding three enzymatic activities, was cloned from Enterococcus faecalis. The mvaE gene encodes a protein with the enzymatic activities of both acetyl-CoA acetyltransferase and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the first and third proteins in the pathway, and the mvaS gene encodes second enzyme in the pathway, HMG-CoA synthase. The mvaE gene was amplified from E. faecalis genomic DNA (ATCC 700802D-5) with an E. coli ribosome binding site and a spacer in front using the following primers:

CF 07-60 (+) Start of mvaE w/ RBS + ATG start codonSacI (SEQ ID NO: 93) 5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTATTA TTG CF 07-62 (−) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 94) 5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTTC TTAAATC

The mvaS gene was amplified from E. faecalis genomic DNA (ATCC 700802D-5) with a RBS and spacer from E. coli in front using the following primers:

CF 07-61 (+) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 95) 5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGA TTGATAAA CF 07-102 (−) End of mvaS gene BglII (SEQ ID NO: 96) 5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT

The PCR fragments were fused together with PCR using the following primers:

CF 07-60 (+) Start of mvaE w/ RBS + ATG start codon SacI (SEQ ID NO: 93) 5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTATT ATTG CF 07-102 (−) End of mvaS gene BglII (SEQ ID NO: 96) 5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT

The fusion PCR fragment was purified using a Qiagen kit and digested with the restriction enzymes Sad and BglII. This digested DNA fragment was gel purified using a Qiagen kit and ligated into the commercially available vector pTrcHis2A, which had been digested with Sad and BglII and gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonies were selected on LA+50 μg/ml carbenicillin plates. A total of six colonies were chosen and grown overnight in LB+50 μg/ml carbenicillin and plasmids were isolated using a Qiagen kit. The plasmids were digested with Sad and BglII to check for inserts and one correct plasmid was sequenced with the following primers:

CF 07-58 (+) Start of mvaE gene (SEQ ID NO: 97) 5′-ATGAAAACAGTAGTTATTATTGATGC CF 07-59 (−) End of mvaE gene (SEQ ID NO: 98) 5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGC CF 07-82 (+) Start of mvaS gene (SEQ ID NO: 99) 5′-ATGACAATTGGGATTGATAAAATTAG CF 07-83 (−) End of mvaS gene (SEQ ID NO: 100) 5′-TTAGTTTCGATAAGAACGAACGGT CF 07-86 (+) Sequence in mvaE (SEQ ID NO: 101) 5′-GAAATAGCCCCATTAGAAGTATC CF 07-87 (+) Sequence in mvaE (SEQ ID NO: 102) 5′-TTGCCAATCATATGATTGAAAATC CF 07-88 (+) Sequence in mvaE (SEQ ID NO: 103) 5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89 (+) Sequence mvaS (SEQ ID NO: 104) 5′-GAAACCTACATCCAATCTTTTGCCC

The plasmid called pTrcHis2AUpperPathway#1 was correct by sequencing and was transformed into the commercially available E. coli strain BL21. Selection was done on LA+50 μg/ml carbenicillin. Two transformants were chosen and grown in LB+50 μg/ml carbenicillin until they reached an OD₆₀₀ of 1.5. Both strains were frozen in a vial at −80° C. in the presence of glycerol. Strains were designated CF 449 for pTrcHis2AUpperPathway#1 in BL21, isolate #1 and CF 450 for pTrcHis2AUpperPathway#1 in BL21, isolate #2. Both clones were found to behave identically when analyzed.

V. Cloning of UpperMVA Pathway into pCL1920

The plasmid pTrcHis2AUpperPathway was digested with the restriction endonuclease SspI to release a fragment containing pTrc-mvaE-mvaS-(His tag)-terminator. In this fragment, the his-tag was not translated. This blunt ended 4.5 kbp fragment was purified from a 1.2% E-gel using the Qiagen Gel Purification kit. A dephosphorylated, blunt ended 4.2 kbp fragment from pCL1920 was prepared by digesting the vector with the restriction endonuclease PvuII, treating with SAP and gel purifying from a 1.2% E-gel using the Qiagen Gel Purification kit. The two fragments were ligated using the Roche Quick Ligation Kit and transformed into TOP10 chemically competent cells. Transformants were selected on LA containing spectinomycin (50 μg/ml). A correct colony was identified by screening for the presence of the insert by PCR. The plasmid was designated pCL PtrcUpperPathway (FIGS. 26 and 27A-27D; SEQ ID NO:12).

VI. Strains Expressing the Combined Upper and Lower Mevalonic Acid Pathways

To obtain a strain with a complete mevalonic acid pathway plus kudzu isoprene synthase, plasmids pTrcKKDyIkISkan and pCLpTrcUpperPathway were both transformed into BL21(λDE3) competent cells (Invitrogen) and transformants were selected on LA containing kanamycin (50 μg/ml) and Spectinomycin (50 μg/ml). The transformants were checked by plasmid prep to ensure that both plasmids were retained in the host. The strain was designated MCM127.

VII. Production of Mevalonic Acid from Glucose in E. coli/pUpperpathway

Single colonies of the BL21/pTrcHis2A-mvaE/mvaS or FM5/p pTrcHis2A-mvaE/mvaS are inoculated into LB+carbenicillin (100 μg/ml) and are grown overnight at 37° C. with shaking at 200 rpm. These cultures were diluted into 50 ml medium in 250 ml baffled flasks to an OD₆₀₀ of 0.1. The medium was TM3+1 or 2% glucose+carbenicillin (100 μg/ml) or TM3+1% glucose+hydrolyzed soy oil+carbenicillin (100 μg/ml) or TM3+biomass (prepared bagasse, corn stover or switchgrass). Cultures were grown at 30° C. with shaking at 200 rpm for approximately 2-3 hours until an OD₆₀₀ of 0.4 was reached. At this point the expression from the mvaE mvaS construct was induced by the addition of IPTG (400 μM). Cultures were incubated for a further 20 or 40 hours with samples taken at 2 hour intervals to 6 hour post induction and then at 24, 36 and 48 hours as needed. Sampling was done by removing 1 ml of culture, measuring the OD₆₀₀, pelleting the cells in a microfuge, removing the supernatant and analyzing it for mevalonic acid.

A 14 liter fermentation of E. coli cells with nucleic acids encoding Enterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase polypeptides produced 22 grams of mevalonic acid with TM3 medium and 2% glucose as the cell medium. A shake flask of these cells produced 2-4 grams of mevalonic acid per liter with LB medium and 1% glucose as the cell culture medium. The production of mevalonic acid in these strains indicated that the MVA pathway was functional in E. coli.

VIII. Production of Isoprene from E. coli BL21 Containing the Upper and Lower MVA Pathway Plus Kudzu Isoprene Synthase.

The following strains were created by transforming in various combinations of plasmids containing the upper and lower MVA pathway and the kudzu isoprene synthase gene as described above and the plasmids containing the idi, dxs, and dxr and isoprene synthase genes described in Example 7. The host cells used were chemically competent BL21(λDE3) and the transformations were done by standard methods. Transformants were selected on L agar containing kanamycin (50 μg/ml) or kanamycin plus spectinomycin (both at a concentration of 50 μg/ml). Plates were grown at 37° C. The resulting strains were designated as follows:

Grown on Kanamycin plus Spectinomycin (50 μg/ml each)

MCM127-pCL Upper MVA+pTrcKKDyIkIS (kan) in BL21(λDE3)

MCM131-pCL1920+pTrcKKDyIkIS (kan) in BL21(λDE3)

MCM125-pCL Upper MVA+pTrcHis2B (kan) in BL21(λDE3)

Grown on Kanamycin (50 μg/ml)

MCM64-pTrcKudzu yIDI DXS (kan) in BL21(λDE3)

MCM50-pTrcKudzu (kan) in BL21(λDE3)

MCM123-pTrcKudzu yIDI DXS DXR (kan) in BL21(λDE3)

The above strains were streaked from freezer stocks to LA+appropriate antibiotic and grown overnight at 37° C. A single colony from each plate was used to inoculate shake flasks (25 ml LB+the appropriate antibiotic). The flasks were incubated at 22° C. overnight with shaking at 200 rpm. The next morning the flasks were transferred to a 37° C. incubator and grown for a further 4.5 hours with shaking at 200 rpm. The 25 ml cultures were centrifuged to pellet the cells and the cells were resuspended in 5 ml LB+the appropriate antibiotic. The cultures were then diluted into 25 ml LB+1% glucose+the appropriate antibiotic to an OD₆₀₀ of 0.1. Two flasks for each strain were set up, one set for induction with IPTG (800 μM) the second set was not induced. The cultures were incubated at 37° C. with shaking at 250 rpm. One set of the cultures were induced after 1.50 hours (immediately following sampling time point 1). At each sampling time point, the OD₆₀₀ was measured and the amount of isoprene determined as described in Example 1. Results are presented in Table 3. The amount of isoprene made is presented as the amount at the peak production for the particular strain.

TABLE 3 Production of isoprene in E. coli strains Strain Isoprene (μg/liter/OD/hr) MCM50   23.8 MCM64 289 MCM125 ND MCM131 Trace MCM127 874 ND: not detected Trace: peak present but not integratable. IX. Analysis of Mevalonic Acid

Mevalonolactone (1.0 g, 7.7 mmol) (CAS#503-48-0) was supplied from Sigma-Aldrich (WI, USA) as a syrup that was dissolved in water (7.7 mL) and was treated with potassium hydroxide (7.7 mmol) in order to generate the potassium salt of mevalonic acid. The conversion to mevalonic acid was confirmed by ¹H NMR analysis. Samples for HPLC analysis were prepared by centrifugation at 14,000 rpm for 5 minutes to remove cells, followed by the addition of a 300 μl aliquot of supernatant to 900 μl of H₂O. Perchloric acid (36 μl of a 70% solution) was then added followed by mixing and cooling on ice for 5 minutes. The samples were then centrifuged again (14,000 rpm for 5 min) and the supernatant transferred to HPLC. Mevalonic acid standards (20, 10, 5, 1 and 0.5 g/L) were prepared in the same fashion. Analysis of mevalonic acid (20 μL injection volume) was performed by HPLC using a BioRad Aminex 87-H+ column (300 mm by 7.0 mm) eluted with 5 mM sulfuric acid at 0.6 mL/min with refractive index (RI) detection. Under these conditions mevalonic acid eluted as the lactone form at 18.5 minutes.

X. Production of Isoprene from E. coli BL21 Containing the Upper MVA Pathway Plus Kudzu Isoprene Synthase

A 15-L scale fermentation of E. coli expressing mevalonic acid pathway polypeptides and Kudzu isoprene synthase was used to produce isoprene from cells in fed-batch culture. This experiment demonstrates that growing cells under glucose limiting conditions resulted in the production of 2.2 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per liter fermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000× modified trace metal solution 1 ml. All of the components were added together and dissolved in diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using the following components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filter sterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the pCL PtrcUpperPathway (FIG. 26) and pTrcKKDyIkIS plasmids. This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37° C. A single colony was inoculated into soytone-yeast extract-glucose medium. After the inoculum grew to OD 1.0 when measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 54 hour fermentation was 3.7 kg. Induction was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The IPTG concentration was brought to 25 μM when the optical density at 550 nm (OD₅₅₀) reached a value of 10. The IPTG concentration was raised to 50 μM when OD₅₅₀ reached 190. IPTG concentration was raised to 100 μM at 38 hours of fermentation. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 54. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 2.2 g/L (FIG. 55). The total amount of isoprene produced during the 54 hour fermentation was 15.9 g, and the time course of production is shown in FIG. 56.

XI. Isoprene Fermentation from E. coli Expressing Genes from the Mevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale

A 15-L scale fermentation of E. coli expressing mevalonic acid pathway polypeptides and Kudzu isoprene synthase was used to produce isoprene from cells in fed-batch culture. This experiment demonstrates that growing cells under glucose limiting conditions resulted in the production of 3.0 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per liter fermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using the following components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filter sterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37° C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached the stationary phase. After this time, the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 59 hour fermentation was 2.2 kg. Induction was achieved by adding IPTG. The IPTG concentration was brought to 25 μM when the optical density at 550 nm (OD₅₅₀) reached a value of 10. The IPTG concentration was raised to 50 μM when OD₅₅₀ reached 190. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 93. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 3.0 g/L (FIG. 94). The total amount of isoprene produced during the 59 hour fermentation was 22.8 g, and the time course of production is shown in FIG. 95. The molar yield of utilized carbon that went into producing isoprene during fermentation was 2.2%. The weight percent yield of isoprene from glucose was 1.0%.

XII. Isoprene Fermentation from E. coli Expressing Genes from the Mevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale

A 15-L scale fermentation of E. coli expressing mevalonic acid pathway polypeptides, Pueraria lobata isoprene synthase, and Kudzu isoprene synthase was used to produce isoprene from cells in fed-batch culture. This experiment demonstrates that growing cells under glucose limiting conditions resulted in the production of 3.3 g/L of isoprene.

i) Construction of pCLPtrcUpperPathwayHGS2

The gene encoding isoprene synthase from Pueraria lobata was PCR-amplified using primers NsiI-RBS-HGS F (CTTGATGCATCCTGCATTCGCCCTTAGGAGG, SEQ ID NO:105) and pTrcR (CCAGGCAAATTCTGTTTTATCAG, SEQ ID NO:106), and pTrcKKDyIkIS as a template. The PCR product thus obtained was restriction-digested with NsiI and PstI and gel-purified. The plasmid pCL PtrcUpperPathway was restriction-digested with PstI and dephosphorylated using rAPid alkaline phosphatase (Roche) according to manufacturer's instructions.

These DNA fragments were ligated together and the ligation reaction was transformed into E. coli Top10 chemically competent cells (Invitrogen), plated on L agar containing spectinomycin (50 μg/ml) and incubated overnight at 37° C. Plasmid DNA was prepared from 6 clones using the Qiaquick Spin Mini-prep kit. The plasmid DNA was digested with restriction enzymes EcoRV and MluI to identify a clone in which the insert had the right orientation (i.e., the gene oriented in the same way as the pTrc promoter).

The resulting correct plasmid was designated pCLPtrcUpperPathwayHGS2. This plasmid was assayed using the headspace assay described herein and found to produce isoprene in E. coli Top10, thus validating the functionality of the gene. The plasmid was transformed into BL21(LDE3) containing pTrcKKDyIkIS to yield the strain BL21/pCLPtrcUpperPathwayHGS2-pTrcKKDyIkIS. This strain has an extra copy of the isoprene synthase compared to the BL21/pCL PtrcUpperMVA and pTrc KKDyIkIS strain (Example 8, part XI). This strain also had increased expression and activity of HMGS compared to the BL21/pCL PtrcUpperMVA and pTrc KKDyIkIS strain used in Example 8, part XI.

ii) Isoprene Fermentation from E. coli Expressing pCLPtrcUpperPathwayHGS2-pTrcKKDyIkIS and Grown in Fed-Batch Culture at the 15-L Scale

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per liter fermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000× modified trace metal solution 1 ml. All of the components were added together and dissolved in diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using the following components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the pCLPtrcUpperPathwayHGS2 and pTrc KKDyIkIS plasmids. This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37° C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0 measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 58 hour fermentation was 2.1 kg. Induction was achieved by adding IPTG. The IPTG concentration was brought to 25 μM when the optical density at 550 nm (OD₅₅₀) reached a value of 9. The IPTG concentration was raised to 50 μM when OD₅₅₀ reached 170. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 104. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 3.3 g/L (FIG. 105). The total amount of isoprene produced during the 58 hour fermentation was 24.5 g and the time course of production is shown in FIG. 106. The molar yield of utilized carbon that went into producing isoprene during fermentation was 2.5%. The weight percent yield of isoprene from glucose was 1.2%. Analysis showed that the activity of the isoprene synthase was increased by approximately 3-4 times that compared to BL21 expressing CL PtrcUpperMVA and pTrc KKDyIkIS plasmids (data not shown).

XIII. Chromosomal Integration of the Lower Mevalonate Pathway in E. coli.

A synthetic operon containing mevalonate kinase, mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, and the IPP isomerase was integrated into the chromosome of E. coli. If desired, expression may be altered by integrating different promoters 5′ of the operon.

Table 4 lists primers used for this experiment.

TABLE 4 Primers MCM78 attTn7 up rev for gcatgctcgagcggccgcTTTTAATCAAACATCCTGCCAA integration construct CTC (SEQ ID NO: 107) MCM79 attTn7 down rev for gatcgaagggcgatcgTGTCACAGTCTGGCGAAACCG integration construct (SEQ ID NO: 108) MCM88 attTn7 up forw for ctgaattctgcagatatcTGTTTTTCCACTCTTCGTTCACTT integration construct T (SEQ ID NO: 109) MCM89 attTn7 down forw for tctagagggcccAAGAAAAATGCCCCGCTTACG (SEQ integration construct ID NO: 110) MCM104 GI1.2 promoter - Gatcgcggccgcgcccttgacgatgccacatcctgagcaaataattcaaccac MVK taattgtgagcggataacacaaggaggaaacagctatgtcattaccgttcttaact tc (SEQ ID NO: 111) MCM105 aspA terminator - Gatcgggccccaagaaaaaaggcacgtcatctgacgtgccttttttatttgtaga yIDI cgcgttgttatagcattcta (SEQ ID NO: 112) MCM120 Forward of attTn7: aaagtagccgaagatgacggtttgtcacatggagttggcaggatgtttgattaaa attTn7 homology, GB agcAATTAACCCTCACTAAAGGGCGG (SEQ ID marker homology NO: 113) MCM127 Rev complement of AGAGTGTTCACCAAAAATAATAACCTTTCCCGG 1.2 GI: GB marker TGCAgaagttaagaacggtaatgacatagctgtttcctccttgtgttatccgct homology(extra long), cacaattagtggttgaattatttgctcaggatgtggcatcgtcaagggcTAAT promoter, RBS, ATG ACGACTCACTATAGGGCTCG (SEQ ID NO: 114) i) Target Vector Construction

The attTn7 site was selected for integration. Regions of homology upstream (attTn7 up) (primers MCM78 and MCM79) and downstream (attTn7 down) (primers MCM88 and MCM89) were amplified by PCR from MG1655 cells. A 50 μL reaction with 1 μL 10 μM primers, 3 μL ddH₂O, 45 μL Invitrogen Platinum PCR Supermix High Fidelity, and a scraped colony of MG1655 was denatured for 2:00 at 94° C., cycled 25 times (2:00 at 94° C., 0:30 at 50° C., and 1:00 at 68° C.), extended for 7:00 at 72° C., and cooled to 4° C. This resulting DNA was cloned into pCR2.1 (Invitrogen) according to the manufacturer's instructions, resulting in plasmids MCM278 (attTn7 up) and MCM252 (attTn7 down). The 832 bp ApaI-PvuI fragment digested and gel purified from MCM252 was cloned into ApaI-PvuI digested and gel purified plasmid pR6K, creating plasmid MCM276. The 825 bp PstI-NotI fragment digested and gel purified from MCM278 was cloned into PstI-NotI digested and gel purified MCM276, creating plasmid MCM281.

ii) Cloning of Lower Pathway and Promoter

MVK-PMK-MVD-IDI genes were amplified from pTrcKKDyIkIS with primers MCM104 and MCM105 using Roche Expand Long PCR System according to the manufacturer's instructions. This product was digested with NotI and ApaI and cloned into MCM281 which had been digested with NotI and ApaI and gel purified. Primers MCM120 and MCM127 were used to amplify CMR cassette from the GeneBridges FRT-gb2-Cm-FRT template DNA using Stratagene Pfu Ultra II. A PCR program of denaturing at 95° C. for 4:00, 5 cycles of 95° C. for 0:20, 55° C. for 0:20, 72° C. for 2:00, 25 cycles of 95° C. for 0:20, 58° C. for 0:20, 72° C. for 2:00, 72° C. for 10:00, and then cooling to 4° C. was used with four 50 μL PCR reactions containing 1 μL˜10 ng/μL template, 1 μL each primer, 1.25 μL 10 mM dNTPs, 5 μL 10× buffer, 1 μL enzyme, and 39.75 μL ddH₂O. Reactions were pooled, purified on a Qiagen PCR cleanup column, and used to electroporate water-washed Pir1 cells containing plasmid MCM296. Electroporation was carried out in 2 mM cuvettes at 2.5V and 200 ohms. Electroporation reactions were recovered in LB for 3 hr at 30° C. Transformant MCM330 was selected on LA with CMP5, Kan50 (FIGS. 107 and 108A-108C; SEQ ID NO:25).

iii) Integration into E. coli Chromosome

Miniprepped DNA (Qiaquick Spin kit) from MCM330 was digested with SnaBI and used to electroporate BL21(DE3) (Novagen) or MG1655 containing GeneBridges plasmid pRedET Carb. Cells were grown at 30° C. to ˜OD1 then induced with 0.4% L-arabinose at 37° C. for 1.5 hours. These cells were washed three times in 4° C. ddH2O before electroporation with 2 μL of DNA. Integrants were selected on L agar with containing chloramphenicol (5 μg/ml) and subsequently confirmed to not grow on L agar+Kanamycin (50 μg/ml). BL21 integrant MCM331 and MG1655 integrant MCM333 were frozen.

iv) Construction of pET24D-Kudzu Encoding Kudzu Isoprene Synthase

The kudzu isoprene synthase gene was subcloned into the pET24d vector (Novagen) from the pCR2.1 vector (Invitrogen). In particular, the kudzu isoprene synthase gene was amplified from the pTrcKudzu template DNA using primers MCM50 5′-GATCATGCAT TCGCCCTTAG GAGGTAAAAA AACATGTGTG CGACCTCTTC TCAATTTACT (SEQ ID NO:52) and MCM53 5′-CGGTCGACGG ATCCCTGCAG TTAGACATAC ATCAGCTG (SEQ ID NO:50). PCR reactions were carried out using Taq DNA Polymerase (Invitrogen), and the resulting PCR product was cloned into pCR2.1-TOPO TA cloning vector (Invitrogen), and transformed into E. coli Top10 chemically competent cells (Invitrogen). Transformants were plated on L agar containing carbenicillin (50 μg/ml) and incubated overnight at 37° C. Five ml Luria Broth cultures containing carbenicillin 50 μg/ml were inoculated with single transformants and grown overnight at 37° C. Five colonies were screened for the correct insert by sequencing of plasmid DNA isolated from 1 ml of liquid culture (Luria Broth) and purified using the QIAprep Spin Mini-prep Kit (Qiagen). The resulting plasmid, designated MCM93, contains the kudzu isoprene synthase coding sequence in a pCR2.1 backbone.

The kudzu coding sequence was removed by restriction endonuclease digestion with PciI and BamH1 (Roche) and gel purified using the QIAquick Gel Extraction kit (Qiagen). The pET24d vector DNA was digested with NcoI and BamHI (Roche), treated with shrimp alkaline phosphatase (Roche), and purified using the QIAprep Spin Mini-prep Kit (Qiagen). The kudzu isoprene synthase fragment was ligated to the NcoI/BamH1 digested pET24d using the Rapid DNA Ligation Kit (Roche) at a 5:1 fragment to vector ratio in a total volume of 20 μl. A portion of the ligation mixture (5 μl) was transformed into E. coli Top 10 chemically competent cells and plated on L agar containing kanamycin (50 μg/ml). The correct transformant was confirmed by sequencing and transformed into chemically competent BL21(λDE3)pLysS cells (Novagen). A single colony was selected after overnight growth at 37° C. on L agar containing kanamycin (50 μg/ml). A map of the resulting plasmid designated as pET24D-Kudzu is shown in FIG. 109. The sequence of pET24D-Kudzu (SEQ ID NO:26) is shown in FIGS. 110A and 110B. Isoprene synthase activity was confirmed using a headspace assay.

v) Production Strains

Strains MCM331 and MCM333 were cotransformed with plasmids pCLPtrcupperpathway and either pTrcKudzu or pETKudzu, resulting in the strains shown in Table 5.

TABLE 5 Production Strains Isoprene Integrated Upper MVA synthase Production Background Lower plasmid plasmid Stain BL21(DE3) MCM331 pCLPtrcUpper pTrcKudzu MCM343 Pathway BL21(DE3) MCM331 pCLPtrcUpper pET24D- MCM335 Pathway Kudzu MG1655 MCM333 pCLPtrcUpper pTrcKudzu MCM345 Pathway vi) Isoprene Fermentation from E. coli Expressing Genes from the Mevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale. Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per liter fermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000× modified trace metal solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using the following components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the gi1.2 integrated lower MVA pathway described above and the pCL PtrcUpperMVA and pTrcKudzu plasmids. This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37° C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached the stationary phase. After this time, the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 57 hour fermentation was 3.9 kg. Induction was achieved by adding IPTG. The IPTG concentration was brought to 100 μM when the carbon dioxide evolution rate reached 100 mmol/L/hr. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 111A. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 1.6 g/L (FIG. 111B). The specific productivity of isoprene over the course of the fermentation is shown in FIG. 111C and peaked at 1.2 mg/OD/hr. The total amount of isoprene produced during the 57 hour fermentation was 16.2 g. The molar yield of utilized carbon that went into producing isoprene during fermentation was 0.9%. The weight percent yield of isoprene from glucose was 0.4%.

XIV. Production of Isoprene from E. coli BL21 Containing the Kudzu Isoprene Synthase Using Glycerol as a Carbon Source

A 15-L scale fermentation of E. coli expressing Kudzu isoprene synthase was used to produce isoprene from cells fed glycerol in fed-batch culture. This experiment demonstrates that growing cells in the presence of glycerol (without glucose) resulted in the production of 2.2 mg/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per liter fermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, and 1000× modified trace metal solution 1 ml. All of the components were added together and dissolved in diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glycerol 5.1 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The medium was generated using the following components per liter fermentation medium: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the pTrcKudzu plasmid. This experiment was carried out to monitor isoprene formation from glycerol at the desired fermentation pH 7.0 and temperature 35° C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LA broth agar plate (with antibiotics) and incubated at 37° C. A single colony was inoculated into soytone-yeast extract-glucose medium and grown at 35° C. After the inoculum grew to OD 1.0, measured at 550 nm, 600 mL was used to inoculate a 7.5-L bioreactor.

Glycerol was fed at an exponential rate until cells reached an optical density at 550 nm (OD₅₅₀) of 153. The total amount of glycerol delivered to the bioreactor during the 36 hour fermentation was 1.7 kg. Other than the glucose in the inoculum, no glucose was added to the bioreactor. Induction was achieved by adding IPTG. The IPTG concentration was brought to 20 μM when the OD₅₅₀ reached a value of 50. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 57. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 2.2 mg/L (FIG. 58). The total amount of isoprene produced during the 54 hour fermentation was 20.9 mg, and the time course of production is shown in FIG. 59.

XV. Isoprene Fermentation from E. coli Expressing Genes from the Mevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale Using Invert Sugar as a Carbon Source

A 15-L scale fermentation of E. coli expressing mevalonic acid pathway polypeptides and Kudzu isoprene synthase was used to produce isoprene from cells fed invert sugar in fed-batch culture. This experiment demonstrates that growing cells in the presence of invert sugar resulted in the production of 2.4 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per liter fermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Invert sugar 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using the following components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in Di H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. This experiment was carried out to monitor isoprene formation from invert sugar at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37° C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

Invert sugar was fed at an exponential rate until cells reached the stationary phase. After this time the invert sugar feed was decreased to meet metabolic demands. The total amount of invert sugar delivered to the bioreactor during the 44 hour fermentation was 2.4 kg. Induction was achieved by adding IPTG. The IPTG concentration was brought to 25 μM when the optical density at 550 nm (OD₅₅₀) reached a value of 9. The IPTG concentration was raised to 50 μM when OD₅₅₀ reached 200. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 96. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 2.4 g/L (FIG. 97). The total amount of isoprene produced during the 44 hour fermentation was 18.4 g and the time course of production is shown in FIG. 98. The molar yield of utilized carbon that went into producing isoprene during fermentation was 1.7%. The weight percent yield of isoprene from glucose was 0.8%.

Example 9 Construction of the Upper and Lower MVA Pathway for Integration into Bacillus subtilis

I. Construction of the Upper MVA Pathway in Bacillus subtilis

The upper pathway from Enterococcus faecalis is integrated into B. subtilis under control of the aprE promoter. The upper pathway consists of two genes; mvaE, which encodes for AACT and HMGR, and mvaS, which encodes for HMGS. The two genes are fused together with a stop codon in between, an RBS site in front of mvaS, and are under the control of the aprE promoter. A terminator is situated after the mvaE gene. The chloramphenicol resistance marker is cloned after the mvaE gene and the construct is integrated at the aprE locus by double cross over using flanking regions of homology.

Four DNA fragments are amplified by PCR such that they contain overhangs that will allowed them to be fused together by a PCR reaction. PCR amplifications are carried out using Herculase polymerase according to manufacturer's instructions.

1. PaprE CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 115) 5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-94 (−) Fuse PaprE to mvaE (SEQ ID NO: 116) 5′-CAATAATAACTACTGTTTTCACTCTTTACCCTCTCCTTTTAA Template: Bacillus subtilis chromosomal DNA 2. mvaE CF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon) (SEQ ID NO: 117) 5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTG CF 07-62 (−) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 94) 5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTTCTTAAATC Template: Enterococcus faecalis chromosomal DNA (from ATCC) 3. mvaS CF 07-61 (+) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 95) 5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGATTGATAAA CF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 118) 5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGT Template: Enterococcus faecalis chromosomal DNA 4. B. amyliquefaciens alkaline serine protease terminator CF 07-123 (+) Fuse the end of mvaS to the terminator (SEQ ID NO: 119) 5′-ACCGTTCGTTCTTATCGAAACTAAAAAAAACCGGCCTTGGCCCCG CF 07-46 (−) End of B. amyliquefaciens terminator BamHI (SEQ ID NO: 58) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC Template: Bacillus amyliquefaciens chromosomal DNA PCR Fusion Reactions 5. Fuse mvaE to mvaS CF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon) (SEQ ID NO: 117) 5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTG CF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 118) 5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGT Template: #2 and 3 from above 6. Fuse mvaE-mvaS to aprE promoter CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 115) 5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 118) 5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGT Template #1 and #4 from above 7. Fuse PaprE-mvaE-mvaS to terminator CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 115) 5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-46 (−) End of B. amyliquefaciens terminator BamHI (SEQ ID NO: 58) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC Template: #4 and #6

The product is digested with restriction endonucleases PstI/BamHI and ligated to pJM102 (Perego, M. 1993. Integrational vectors for genetic manipulation in Bacillus subtilis, p. 615-624. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics. American Society for Microbiology, Washington, D.C.) which is digested with PstI/BamHI. The ligation is transformed into E. coli TOP 10 chemically competent cells and transformants are selected on LA containing carbenicillin (50 μg/ml). The correct plasmid is identified by sequencing and is designated pJMUpperpathway2 (FIGS. 50 and 51). Purified plasmid DNA is transformed into Bacillus subtilis aprEnprE Pxyl-comK and transformants are selected on L agar containing chloramphenicol (5 μg/ml). A correct colony is selected and is plated sequentially on L agar containing chloramphenicol 10, 15 and 25 μg/ml to amplify the number of copies of the cassette containing the upper pathway.

The resulting strain is tested for mevalonic acid production by growing in LB containing 1% glucose and 1%. Cultures are analyzed by GC for the production of mevalonic acid.

This strain is used subsequently as a host for the integration of the lower mevalonic acid pathway.

The following primers are used to sequence the various constructs above.

Sequencing Primers:

CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 115) 5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-58 (+) Start of mvaE gene (SEQ ID NO: 97) 5′-ATGAAAACAGTAGTTATTATTGATGC CF 07-59 (−) End of mvaE gene (SEQ ID NO: 98) 5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGC CF 07-82 (+) Start of mvaS gene (SEQ ID NO: 99) 5′-ATGACAATTGGGATTGATAAAATTAG CF 07-83 (−) End of mvaS gene (SEQ ID NO: 100) 5′-TTAGTTTCGATAAGAACGAACGGT CF 07-86 (+) Sequence in mvaE (SEQ ID NO: 101) 5′-GAAATAGCCCCATTAGAAGTATC CF 07-87 (+) Sequence in mvaE (SEQ ID NO: 102) 5′-TTGCCAATCATATGATTGAAAATC CF 07-88 (+) Sequence in mvaE (SEQ ID NO: 103) 5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89 (+) Sequence mvaS (SEQ ID NO: 104) 5′-GAAACCTACATCCAATCTTTTGCCC

Transformants are selected on LA containing chloramphenicol at a concentration of 5 μg/ml. One colony is confirmed to have the correct integration by sequencing and is plated on LA containing increasing concentrations of chloramphenicol over several days, to a final level of 25 μg/ml. This results in amplification of the cassette containing the genes of interest. The resulting strain is designated CF 455: pJMupperpathway#1 X Bacillus subtilis aprEnprE Pxyl comK (amplified to grow on LA containing chloramphenicol 25 μg/ml).

II. Construction of the Lower MVA Pathway in Bacillus subtilis

The lower MVA pathway, consisting of the genes mvk1, pmk, mpd and idi are combined in a cassette consisting of flanking DNA regions from the nprE region of the B. subtilis chromosome (site of integration), the aprE promoter, and the spectinomycin resistance marker (see FIGS. 28 and 29; SEQ ID NO:13). This cassette is synthesized by DNA2.0 and is integrated into the chromosome of B. subtilis containing the upper MVA pathway integrated at the aprE locus. The kudzu isoprene synthase gene is expressed from the replicating plasmid described in Example 4 and is transformed into the strain with both upper and lower pathways integrated.

Example 10 Exemplary Isoprene Compositions and Methods of Making Them

I. Compositional Analysis of Fermentation Off-Gas Containing Isoprene

A 14 L scale fermentation was performed with a recombinant E. coli BL21 (DE3) strain containing two plasmids (pCL upperMev; pTrcKKDyIkIS encoding the full mevalonate pathway for isoprenoid precursor biosynthesis, an isoprenyl pyrophosphate isomerase from yeast, and an isoprene synthase from Kudzu. Fermentation off-gas from the 14 L tank was collected into 20 mL headspace vials at around the time of peak isoprene productivity (27.9 hours elapsed fermentation time, “EFT”) and analyzed by headspace GC/MS for volatile components.

Headspace analysis was performed with an Agilent 6890 GC/MS system fitted with an Agilent HP-5MS GC/MS column (30 m×250 μm; 0.25 μm film thickness). A combiPAL autoinjector was used for sampling 500 μL aliquots from 20 mL headspace vials. The GC/MS method utilized helium as the carrier gas at a flow of 1 mL/min. The injection port was held at 250° C. with a split ratio of 50:1. The oven temperature was held at 37° C. for an initial 2 minute period, followed an increase to 237° C. at a rate of 25° C./min for a total method time of 10 minutes. The Agilent 5793N mass selective detector scanned from m/z 29 to m/z 300. The limit of detection of this system is approximately 0.1 μg/L_(gas) or approximately 0.1 ppm. If desired, more sensitive equipment with a lower limit of detection may be used.

The off-gas consisted of 99.925% (v/v) permanent gases (N₂, CO₂ and O₂), approximately 0.075% isoprene (2-methyl-1,3-butadiene) (˜750 ppmv, 2100 μg/L) and minor amounts (<50 ppmv) of ethanol, acetone, and two C5 prenyl alcohols. The amount of water vapor was not determined but was estimated to be equal to the equilibrium vapor pressure at 0° C. The composition of the volatile organic fraction was determined by integration of the area under the peaks in the GC/MS chromatogram (FIGS. 86A and 86B) and is listed in Table 6. Calibration curves for ethanol and acetone standards enabled the conversion of GC area to gas phase concentration in units of μg/L using standard methods.

TABLE 6 Composition of volatile organic components in fermentation off-gas. The off-gas was analyzed at the 27.9 hour time point of a fermentation using an E. coli BL21 (DE3) strain expressing a heterologous mevalonate pathway, an isoprenyl pyrophosphate isomerase from yeast, and an isoprene synthase from Kudzu. Compound RT (min) GC area Area % Conc. (μg/L) Ethanol 1.669 239005 0.84 62 +/− 6 Acetone 1.703 288352 1.02 42 +/− 4 Isoprene (2-methyl- 1.829 27764544 97.81 2000 +/− 200 1,3-butadiene) 3-methyl-3-buten-1-ol 3.493 35060 0.12 <10 3-methyl-2-buten-1-ol 4.116 58153 0.20 <10 II. Measurement of Trace Volatile Organic Compounds (VOCs) Co-Produced with Isoprene During Fermentation of a Recombinant E. coli Strain

A 14 L scale fermentation was performed with a recombinant E. coli BL21 (DE3) strain containing two plasmids (pCL upperMev; pTrcKKDyIkIS) encoding the full mevalonate pathway for isoprenoid precursor biosynthesis, an isoprenyl pyrophosphate isomerase from yeast, and an isoprene synthase from Kudzu.

Fermentation off-gas was passed through cooled headspace vials in order to concentrate and identify trace volatile organic components. The off-gas from this fermentation was sampled at a rate of 1 L/min for 10 minutes through a 20 mL headspace vial packed with quartz wool (2 g) and cooled to −78° C. with dry ice. The vial was recapped with a fresh vial cap and analyzed by headspace GC/MS for trapped VOCs using the conditions described in Example 10, part I. The ratios of compounds observed in FIGS. 87A-87D are a combination of overall level in the fermentation off-gas, the relative vapor pressure at −78° C., and the detector response of the mass spectrometer. For example, the low level of isoprene relative to oxygenated volatiles (e.g., acetone and ethanol) is a function of the high volatility of this material such that it does not accumulate in the headspace vial at −78° C.

The presence of many of these compounds is unique to isoprene compositions derived from biological sources. The results are depicted in FIGS. 87A-87D and summarized in Tables 7A and 7B.

TABLE 7A Trace volatiles present in off-gas produced by E. coli BL21 (DE3) (pCL upperMev; pTrcKKDyIkIS) following cryo-trapping at −78° C. RT GC Compound (min) Area1 Area %2 Ratio %3 Acetaldehyde 1.542 4019861 4.841 40.14 Ethanol 1.634 10553620 12.708 105.39 Acetone 1.727 7236323 8.714 72.26 2-methyl-1,3-butadiene 1.777 10013714 12.058 100.00 1-propanol 1.987 163574 0.197 1.63 Diacetyl 2.156 221078 0.266 2.21 2-methyl-3-buten-2-ol 2.316 902735 1.087 9.01 2-methyl-1-propanol 2.451 446387 0.538 4.46 3-methyl-1-butanal 2.7 165162 0.199 1.65 1-butanol 2.791 231738 0.279 2.31 3-methyl-3-buten-1-ol 3.514 14851860 17.884 148.32 3-methyl-1-butanol 3.557 8458483 10.185 84.47 3-methyl-2-buten-1-ol 4.042 18201341 21.917 181.76 3-methyl-2-butenal 4.153 1837273 2.212 18.35 3-methylbutyl acetate 5.197 196136 0.236 1.96 3-methyl-3-buten-1-yl 5.284 652132 0.785 6.51 acetate 2-heptanone 5.348 67224 0.081 0.67 2,5-dimethylpyrazine 5.591 58029 0.070 0.58 3-methyl-2-buten-1-yl 5.676 1686507 2.031 16.84 acetate 6-methyl-5-hepten-2-one 6.307 101797 0.123 1.02 2,4,5-trimethylpyridine 6.39 68477 0.082 0.68 2,3,5-trimethylpyrazine 6.485 30420 0.037 0.30 (E)-3,7-dimethyl-1,3,6- 6.766 848928 1.022 8.48 octatriene (Z)-3,7-dimethyl-1,3,6- 6.864 448810 0.540 4.48 octatriene 3-methyl-2-buten-1-yl 7.294 105356 0.127 1.05 butyrate Citronellal 7.756 208092 0.251 2.08 2,3-cycloheptenolpyridine 8.98 1119947 1.349 11.18 1GC area is the uncorrected area under the peak corresponding to the listed compound. 2Area % is the peak area expressed as a % relative to the total peak area of all compounds. 3Ratio % is the peak area expressed as a % relative to the peak area of 2-methyl-1,3-butadiene.

TABLE 7B Trace volatiles present in off-gas produced by E. coli BL21 (DE3) (pCL upperMev; pTrcKKDyIkIS) following cryo-trapping at −196° C. RT GC Ratio Compound (min) Area1 Area %2 %3 Acetaldehyde 1.54 1655710 0.276 0.33 Methanethiol 1.584 173620 0.029 0.03 Ethanol 1.631 10259680 1.707 2.03 Acetone 1.722 73089100 12.164 14.43 2-methyl-1,3-butadiene 1.771 506349429 84.269 100.00 methyl acetate 1.852 320112 0.053 0.06 1-propanol 1.983 156752 0.026 0.03 Diacetyl 2.148 67635 0.011 0.01 2-butanone 2.216 254364 0.042 0.05 2-methyl-3-buten-2-ol 2.312 684708 0.114 0.14 ethyl acetate 2.345 2226391 0.371 0.44 2-methyl-1-propanol 2.451 187719 0.031 0.04 3-methyl-1-butanal 2.696 115723 0.019 0.02 3-methyl-2-butanone 2.751 116861 0.019 0.02 1-butanol 2.792 54555 0.009 0.01 2-pentanone 3.034 66520 0.011 0.01 3-methyl-3-buten-1-ol 3.516 1123520 0.187 0.22 3-methyl-1-butanol 3.561 572836 0.095 0.11 ethyl isobutyrate 3.861 142056 0.024 0.03 3-methyl-2-buten-1-ol 4.048 302558 0.050 0.06 3-methyl-2-butenal 4.152 585690 0.097 0.12 butyl acetate 4.502 29665 0.005 0.01 3-methylbutyl acetate 5.194 271797 0.045 0.05 3-methyl-3-buten-1-yl acetate 5.281 705366 0.117 0.14 3-methyl-2-buten-1-yl acetate 5.675 815186 0.136 0.16 (E)-3,7-dimethyl-1,3,6- 6.766 207061 0.034 0.04 octatriene (Z)-3,7-dimethyl-1,3,6- 6.863 94294 0.016 0.02 octatriene 2,3-cycloheptenolpyridine 8.983 135104 0.022 0.03 1GC area is the uncorrected area under the peak corresponding to the listed compound. 2Area % is the peak area expressed as a % relative to the total peak area of all compounds. 3Ratio % is the peak area expressed as a % relative to the peak area of 2-methyl-1,3-butadiene. III. Absence of C5 Hydrocarbon Isomers in Isoprene Derived from Fermentation.

Cryo-trapping of isoprene present in fermentation off-gas was performed using a 2 mL headspace vial cooled in liquid nitrogen. The off-gas (1 L/min) was first passed through a 20 mL vial containing sodium hydroxide pellets in order to minimize the accumulation of ice and solid CO₂ in the 2 mL vial (−196° C.). Approximately 10 L of off-gas was passed through the vial, after which it was allowed to warm to −78° C. with venting, followed by resealing with a fresh vial cap and analysis by GC/MS.

GC/MS headspace analysis was performed with an Agilent 6890 GC/MS system using a 100 μL gas tight syringe in headspace mode. A Zebron ZB-624 GC/MS column (30 m×250 μm; 1.40 μm film thickness) was used for separation of analytes. The GC autoinjector was fitted with a gas-tight 100 μL syringe, and the needle height was adjusted to allow the injection of a 50 μL headspace sample from a 2 mL GC vial. The GC/MS method utilized helium as the carrier gas at a flow of 1 mL/min. The injection port was held at 200° C. with a split ratio of 20:1. The oven temperature was held at 37° C. for the 5 minute duration of the analysis. The Agilent 5793N mass selective detector was run in single ion monitoring (SIM) mode on m/z 55, 66, 67 and 70. Under these conditions, isoprene was observed to elute at 2.966 minutes (FIG. 88B). A standard of petroleum derived isoprene (Sigma-Aldrich) was also analyzed using this method and was found to contain additional C5 hydrocarbon isomers, which eluted shortly before or after the main peak and were quantified based on corrected GC area (FIG. 88A).

TABLE 8A GC/MS analysis of petroleum-derived isoprene Area % of total RT C5 Compound (min) GC area hydrocarbons 2-methyl-1-butene 2.689 18.2 × 103 0.017% (Z)-2-pentene 2.835 10.6 × 104 0.101% Isoprene 2.966 10.4 × 107 99.869% 1,3- 3.297 12.8 × 103 0.012% cyclopentadiene (CPD)

TABLE 8B GC/MS analysis of fermentation-derived isoprene (% total C5 hydrocarbons) Corrected GC % of total C5 Compound RT (min) Area hydrocarbons Isoprene 2.966 8.1 × 107 100%

In a separate experiment, a standard mixture of C5 hydrocarbons was analyzed to determine if the detector response was the same for each of the compounds. The compounds were 2-methyl-1-butene, 2-methyl-1,3-butadiene, (E)-2-pentene, (Z)-2-pentene and (E)-1,3-pentadiene. In this case, the analysis was performed on an Agilent DB-Petro column (100 m×0.25 mm, 0.50 μm film thickness) held at 50° C. for 15 minutes. The GC/MS method utilized helium as the carrier gas at a flow of 1 mL/min. The injection port was held at 200° C. with a split ratio of 50:1. The Agilent 5793N mass selective detector was run in full scan mode from m/z 19 to m/z 250. Under these conditions, a 100 μg/L concentration of each standard produced the same detector response within experimental error.

IV. Compositions Comprising Isoprene Adsorbed to a Solid Phase.

Biologically-produced isoprene was adsorbed to activated carbon resulting in a solid phase containing 50 to 99.9% carbon, 0.1% to 50% isoprene, 0.01% to 5% water, and minor amounts (<0.1%) of other volatile organic components.

Fermentation off-gas was run through a copper condensation coil held at 0° C., followed by a granulated silica desiccant filter in order to remove water vapor. The dehumidified off-gas was then run through carbon containing filters (Koby Jr, Koby Filters, MA) to the point at which breakthrough of isoprene was detected in the filter exhaust by GC/MS. The amount of isoprene adsorbed to the cartridge can be determined indirectly by calculating the concentration in the off-gas, the overall flow rate and the percent breakthrough over the collection period. Alternately the adsorbed isoprene can be recovered from the filters by thermal, vacuum, or solvent-mediated desorption.

V. Collection and Analysis of Condensed Isoprene.

Fermentation off-gas is dehumidified, and the CO₂ removed by filtration through a suitable adsorbent (e.g., ascarite). The resulting off-gas stream is then run through a liquid nitrogen-cooled condenser in order to condense the VOCs in the stream. The collection vessel contains t-butyl catechol to inhibit the resulting isoprene condensate. The condensate is analyzed by GC/MS and NMR in order to determine purity using standard methods, such as those described herein.

VI. Production of Prenyl Alcohols by Fermentation

Analysis of off-gas from an E. coli BL21 (DE3) strain expressing a Kudzu isoprene synthase revealed the presence of both isoprene and 3-methyl-3-buten-1-ol (isoprenol). The levels of the two compounds in the fermentation off-gas over the fermentation are shown in FIG. 89 as determined by headspace GC/MS. Levels of isoprenol (3-methyl-3-buten-1-ol, 3-MBA) attained was nearly 10 μg/L_(offgas) in this experiment. Additional experiments produced levels of approximately 20 μg/L_(offgas) in the fermentation off-gas.

Example 11 The De-Coupling of Growth and Production of Isoprene in E. coli Expressing Genes from the Mevalonic Acid Pathway and Fermented in a Fed-Batch Culture

Example 11 illustrates the de-coupling of cell growth from mevalonic acid and isoprene production.

I. Fermentation Conditions

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per liter fermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000× modified trace metal solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using the following components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filter sterilized with a 0.22 micron filter.

Fermentation was performed with E. coli cells containing the pTrcHis2AUpperPathway (also called pTrcUpperMVA, FIGS. 91 and 92A-92C; SEQ ID NO:23) (50 μg/ml carbenicillin) or the pCL PtrcUpperMVA (also called pCL PtrcUpperPathway (FIG. 26)) (50 μg/ml spectinomycin) plasmids. For experiments in which isoprene was produced, the E. coli cells also contained the pTrc KKDyIkIS (50 μg/ml kanamycin) plasmid. These experiments were carried out to monitor mevalonic acid or isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of an E. coli strain taken from a frozen vial was streaked onto an LA broth agar plate (with antibiotics) and incubated at 37° C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to optical density 1.0 when measured at 550 nm, it was used to inoculate the bioreactor.

Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. Induction was achieved by adding IPTG. The mevalonic acid concentration in fermentation broth was determined by applying perchloric acid (Sigma-Aldrich #244252) treated samples (0.3 M incubated at 4° C. for 5 minutes) to an organic acids HPLC column (BioRad #125-0140). The concentration was determined by comparing the broth mevalonic acid peak size to a calibration curve generated from mevalonolacetone (Sigma-Aldrich #M4667) treated with perchloric acid to form D,L-mevalonate. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer is defined as the amount of isoprene produced per liter of fermentation broth.

II. Mevalonic Acid Production from E. coli BL21 (DE3) Cells Expressing the pTrcUpperMVA Plasmid at a 150-L Scale

BL21 (DE3) cells that were grown on a plate as explained above in Example 11, part I were inoculated into a flask containing 45 mL of tryptone-yeast extract medium and incubated at 30° C. with shaking at 170 rpm for 5 hours. This solution was transferred to a 5-L bioreactor of tryptone-yeast extract medium, and the cells were grown at 30° C. and 27.5 rpm until the culture reached an OD₅₅₀ of 1.0. The 5 L of inoculum was seeded into a 150-L bioreactor containing 45-kg of medium. The IPTG concentration was brought to 1.1 mM when the OD₅₅₀ reached a value of 10. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 60A. The mevalonic acid titer increased over the course of the fermentation to a final value of 61.3 g/L (FIG. 60B). The specific productivity profile throughout the fermentation is shown in FIG. 60C and a comparison to FIG. 60A illustrates the de-coupling of growth and mevalonic acid production. The total amount of mevalonic acid produced during the 52.5 hour fermentation was 4.0 kg from 14.1 kg of utilized glucose. The molar yield of utilized carbon that went into producing mevalonic acid during fermentation was 34.2%.

III. Mevalonic Acid Production from E. coli BL21 (DE3) Cells Expressing the pTrcUpperMVA Plasmid at a 15-L Scale

BL21 (DE3) cells that were grown on a plate as explained above in Example 11, part I were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG concentration was brought to 1.0 mM when the OD₅₅₀ reached a value of 10. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 61A. The mevalonic acid titer increased over the course of the fermentation to a final value of 53.9 g/L (FIG. 61B). The specific productivity profile throughout the fermentation is shown in FIG. 61C and a comparison to FIG. 61A illustrates the de-coupling of growth and mevalonic acid production. The total amount of mevalonic acid produced during the 46.6 hour fermentation was 491 g from 2.1 kg of utilized glucose. The molar yield of utilized carbon that went into producing mevalonic acid during fermentation was 28.8%.

IV. Mevalonic Acid Production from E. coli FM5 Cells Expressing the pTrcUpperMVA Plasmid at a 15-L Scale

FM5 cells that were grown on a plate as explained above in Example 11, part I were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG concentration was brought to 1.0 mM when the OD₅₅₀ reached a value of 30. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 62A. The mevalonic acid titer increased over the course of the fermentation to a final value of 23.7 g/L (FIG. 62B). The specific productivity profile throughout the fermentation is shown in FIG. 62C and a comparison to FIG. 62A illustrates the de-coupling of growth and mevalonic acid production. The total amount of mevalonic acid produced during the 51.2 hour fermentation was 140 g from 1.1 kg of utilized glucose. The molar yield of utilized carbon that went into producing mevalonic acid during fermentation was 15.2%.

V. Isoprene Production from E. coli BL21 (DE3) Cells Expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

BL21 (DE3) cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids that were grown on a plate as explained above in Example 11, part I were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG concentration was brought to 25 μM when the OD₅₅₀ reached a value of 10. The IPTG concentration was raised to 50 μM when OD₅₅₀ reached 190. The IPTG concentration was raised to 100 μM at 38 hours of fermentation. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 63A. The isoprene titer increased over the course of the fermentation to a final value of 2.2 g/L broth (FIG. 63B). The specific productivity profile throughout the fermentation is shown in FIG. 63C and a comparison to FIG. 63A illustrates the de-coupling of growth and isoprene production. The total amount of isoprene produced during the 54.4 hour fermentation was 15.9 g from 2.3 kg of utilized glucose. The molar yield of utilized carbon that went into producing isoprene during fermentation was 1.53%.

VI. Isoprene Production from E. coli BL21 (DE3) Tuner Cells Expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

BL21 (DE3) tuner cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids that were grown on a plate as explained above in Example 11, part I were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG concentration was brought to 26 μM when the OD₅₅₀ reached a value of 10. The IPTG concentration was raised to 50 μM when OD₅₅₀ reached 175. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 64A. The isoprene titer increased over the course of the fermentation to a final value of 1.3 g/L broth (FIG. 64B). The specific productivity profile throughout the fermentation is shown in FIG. 64C and a comparison to FIG. 64A illustrates the de-coupling of growth and isoprene production. The total amount of isoprene produced during the 48.6 hour fermentation was 9.9 g from 1.6 kg of utilized glucose. The molar yield of utilized carbon that went into producing isoprene during fermentation was 1.34%.

VII. Isoprene Production from E. coli MG1655 Cells Expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

MG1655 cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids that were grown on a plate as explained above in Example 11, part I were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG concentration was brought to 24 μM when the OD₅₅₀ reached a value of 45. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 65A. The isoprene titer increased over the course of the fermentation to a final value of 393 mg/L broth (FIG. 65B). The specific productivity profile throughout the fermentation is shown in FIG. 65C and a comparison to FIG. 65A illustrates the de-coupling of growth and isoprene production. The total amount of isoprene produced during the 67.4 hour fermentation was 2.2 g from 520 g of utilized glucose. The molar yield of utilized carbon that went into producing isoprene during fermentation was 0.92%.

VIII. Isoprene Production from E. coli MG1655ack-pta Cells Expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

MG1655ack-pta cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids that were grown on a plate as explained above in Example 11, part I were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG concentration was brought to 30 μM when the OD₅₅₀ reached a value of 10. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 66A. The isoprene titer increased over the course of the fermentation to a final value of 368 mg/L broth (FIG. 66B). The specific productivity profile throughout the fermentation is shown in FIG. 66C and a comparison to FIG. 66A illustrates the de-coupling of growth and isoprene production. The total amount of isoprene produced during the 56.7 hour fermentation was 1.8 g from 531 g of utilized glucose. The molar yield of utilized carbon that went into producing isoprene during fermentation was 0.73%.

IX. Isoprene Production from E. coli FM5 Cells Expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

FM5 cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids that were grown on a plate as explained above in Example 11, part I were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG concentration was brought to 27 μM when the OD₅₅₀ reached a value of 15. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 67A. The isoprene titer increased over the course of the fermentation to a final value of 235 mg/L broth (FIG. 67B). The specific productivity profile throughout the fermentation is shown in FIG. 67C and a comparison to FIG. 67A illustrates the de-coupling of growth and isoprene production. The total amount of isoprene produced during the 52.3 hour fermentation was 1.4 g from 948 g of utilized glucose. The molar yield of utilized carbon that went into producing isoprene during fermentation was 0.32%.

Example 12 Production of Isoprene During the Exponential Growth Phase of E. coli Expressing Genes from the Mevalonic Acid Pathway and Fermented in a Fed-Batch Culture

Example 12 illustrates the production of isoprene during the exponential growth phase of cells.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per liter fermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000× modified trace metal solution 1 ml. All of the components were added together and dissolved in diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using the following components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in Di H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with ATCC 11303 E. coli cells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37° C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 50 hour fermentation was 2.0 kg. Induction was achieved by adding IPTG. The IPTG concentration was brought to 25 μM when the optical density at 550 nm (OD₅₅₀) reached a value of 10. The IPTG concentration was raised to 50 μM when OD₅₅₀ reached 190. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 99. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 1.4 g/L (FIG. 100). The total amount of isoprene produced during the 50 hour fermentation was 10.0 g. The profile of the isoprene specific productivity over time within the bioreactor is shown in FIG. 101. The molar yield of utilized carbon that contributed to producing isoprene during fermentation was 1.1%. The weight percent yield of isoprene from glucose was 0.5%.

Example 13 Flammability Modeling and Testing of Isoprene

I. Summary of Flammability Modeling and Testing of Isoprene

Flammability modeling and experiments were performed for various hydrocarbon/oxygen/nitrogen/water/carbon dioxide mixtures. This modeling and experimental tested was aimed at defining isoprene and oxygen/nitrogen flammability curves under specified steam and carbon monoxide concentrations at a fixed pressure and temperature. A matrix of the model conditions is shown in Table 9, and a matrix of the experiments performed is shown in Table 5.

TABLE 9 Summary of Modeled Isoprene Flammability Carbon Steam Dioxide Isoprene Oxygen Temperature Pressure Concentration Concentration Concentration Concentration Series (° C.) (psig) (wt %) (wt. %) (vol. %) (vol. %) A 40 0 0 0 Varying Varying B 40 0 4 0 Varying Varying C 40 0 0 5 Varying Varying D 40 0 0 10 Varying Varying E 40 0 0 15 Varying Varying F 40 0 0 20 Varying Varying G 40 0 0 30 Varying Varying

TABLE 10 Summary of Isoprene Flammability Tests Steam Isoprene Oxygen Temperature Pressure Concentration Concentration Concentration Series Number (° C.) (psig) (vol. %) (vol. %) (vol. %) 1 40 0 0 Varying Varying 2 40 0 4 Varying Varying II. Description of Calculated Adiabatic Flame Temperature (CAFT) Model

Calculated adiabatic flame temperatures (CAFT) along with a selected limit flame temperature for combustion propagation were used to determine the flammability envelope for isoprene. The computer program used in this study to calculate the flame temperatures is the NASA Glenn Research Center CEA (Chemical Equilibrium with Applications) software.

There are five steps involved in determining the flammability envelope using an adiabatic flame temperature model for a homogeneous combustion mechanism (where both the fuel and oxidant are in the gaseous state): selection of the desired reactants, selection of the test condition, selection of the limit flame temperature, modification of the reactants, and construction of a flammability envelope from calculations.

In this first step, selection of desired reactants, a decision must be made as to the reactant species that will be present in the system and the quantities of each. In many cases the computer programs used for the calculations have a list of reactant and product species. If any of the data for the species to be studied are not found in the program, they may be obtained from other sources such as the JANAF tables or from the internet. In this current model data for water, nitrogen, oxygen and carbon dioxide were present in the program database. The program database did not have isoprene as a species; therefore the thermodynamic properties were incorporated manually.

The next step is to decide whether the initial pressure and temperature conditions that the combustion process is taking place in. In this model the pressure was 1 atmosphere (absolute) and the temperature was 40° C., the boiling point of isoprene.

The limit flame temperature for combustion can be either selected based on theoretical principles or determined experimentally. Each method has its own limitations.

Based on prior studies, the limit flame temperatures of hydrocarbons fall in the range of 1000 K to 1500 K. For this model, the value of 1500 K was selected. This is the temperature at which the reaction of carbon monoxide to carbon dioxide (a highly exothermic reaction and constitutes a significant proportion of the flame energy) becomes self sustaining

Once the limit flame temperature has been decided upon, model calculations are performed on the given reactant mixture (species concentrations) and the adiabatic flame temperature is determined. Flame propagation is considered to have occurred only if the temperature is greater than the limit flame temperature. The reactant mixture composition is then modified to create data sets for propagation and non-propagation mixtures.

This type of model shows good agreement with the experimentally determined flammability limits. Regions outside the derived envelope are nonflammable and regions within it are flammable. The shape of the envelope forms a nose. The nose of the envelope is related to the limiting oxygen concentration (LOC) for gaseous fuels.

III. Results from Calculated Adiabatic Flame Temperature (CAFT) Model

Plotted in FIGS. 68 through 74 are the CAFT model results for Series A to G, respectively. The figures plot the calculated adiabatic flame temperature (using the NASA CEA program) as a function of fuel concentration (by weight) for several oxygen/nitrogen ratios (by weight). The parts of the curve that are above 1500 K, the selected limit flame temperature, contain fuel levels sufficient for flame propagation. The results may be difficult to interpret in the form presented in FIGS. 68 through 74. Additionally, the current form is not conducive to comparison with experimental data which is generally presented in terms of volume percent.

Using Series A as an example the data in FIG. 68 can be plotted in the form of a traditional flammability envelope. Using FIG. 68 and reading across the 1500 K temperature line on the ordinate one can determine the fuel concentration for this limit flame temperature by dropping a tangent to the abscissa for each curve (oxygen to nitrogen ratio) that it intersects. These values can then be tabulated as weight percent of fuel for a given weight percent of oxidizer (FIG. 75A). Then knowing the composition of the fuel (100 wt. % isoprene) and the composition of the oxidizer (relative content of water, oxygen and nitrogen) molar quantities can be established.

From these molar quantities percentage volume concentrations can be calculated. The concentrations in terms of volume percent can then be plotted to generate a flammability envelope (FIG. 75B). The area bounded by the envelope is the explosible range and the area excluded is the non-explosible range. The “nose” of the envelope is the limiting oxygen concentration. FIGS. 76A and 76B contain the calculated volume concentrations for the flammability envelope for Series B generated from data presented in FIG. 69. A similar approach can be used on data presented in FIGS. 70-74.

IV. Flammability Testing Experimental Equipment and Procedure

Flammability testing was conducted in a 4 liter high pressure vessel. The vessel was cylindrical in shape with an inner diameter of 6″ and an internal height of 8.625″. The temperature of the vessel (and the gases inside) was maintained using external heaters that were controlled by a PID controller. To prevent heat losses, ceramic wool and reflective insulation were wrapped around the pressure vessel. Type K thermocouples were used the measure the temperature of the gas space as well as the temperature of the vessel itself. FIG. 77 illustrates the test vessel.

Before a test was ran, the vessel was evacuated and purged with nitrogen to ensure that any gases from previous tests were removed. A vacuum was then pulled on the vessel. The pressure after this had been done was typically around 0.06 bar(a). Due to the nitrogen purging, the gas responsible for this initial pressure was assumed to be nitrogen. Using partial pressures, water, isoprene, nitrogen, and oxygen were then added in the appropriate amounts to achieve the test conditions in question. A magnetically driven mixing fan within the vessel ensured mixing of the gaseous contents. The gases were allowed to mix for about 2 minutes with the fan being turned off approximately 1 minute prior to ignition.

The igniter was comprised of a 1.5 ohm nicrome coil and an AC voltage source on a timer circuit. Using an oscilloscope, it was determined that 34.4 VAC were delivered to the igniter for 3.2 seconds. A maximum current of 3.8 amps occurred approximately halfway into the ignition cycle. Thus, the maximum power was 131 W and the total energy provided over the ignition cycle was approximately 210 J.

Deflagration data was acquired using a variable reluctance Validyne DP215 pressure transducer connected to a data acquisition system. A gas mixture was considered to have deflagrated if the pressure rise was greater than or equal to 5%.

V. Results of Flammability Testing

The first experimental series (Series 1) was run at 40° C. and 0 psig with no steam. Running tests at varying concentrations of isoprene and oxygen produced the flammability curve shown in FIG. 78A. The data points shown in this curve are only those that border the curve. A detailed list of all the data points taken for this series is shown in FIGS. 80A and 80B.

FIG. 78B summarizes the explosibility data points shown in FIG. 78A. FIG. 78C is a comparison of the experimental data with the CAFT model predicted flammability envelope. The model agrees very well with the experimental data. Discrepancies may be due to the non-adiabatic nature of the test chamber and limitations of the model. The model looks at an infinite time horizon for the oxidation reaction and does not take into consideration any reaction kinetic limitation.

Additionally, the model is limited by the number of equilibrium chemical species that are in its database and thus may not properly predict pyrolytic species. Also, the flammability envelope developed by the model uses one value for a limit flame temperature (1500K). The limit flame temperature can be a range of values from 1,000K to 1,500K depending on the reacting chemical species. The complex nature of pyrolytic chemical species formed at fuel concentrations above the stoichiometric fuel/oxidizer level is one reason why the model may not accurately predict the upper flammable limit for this system.

The second experimental series (Series 2) was run at 40° C. and 0 psig with a fixed steam concentration of 4%. Running tests at varying concentrations of isoprene and oxygen produced the flammability curve shown in FIG. 79A. The data points shown in this curve are only those that border the curve. A detailed list of all the data points taken for this series is shown in FIG. 81. Due to the similarity between the data in Series 1 only the key points of lower flammable limit, limiting oxygen concentration, and upper flammable limits were tested. The addition of 4% steam to the test mixture did not significantly change the key limits of the flammability envelope. It should be noted that higher concentrations of steam/water and or other inertants may influence the flammability envelope.

FIG. 79B summarizes the explosibility data points shown in FIG. 79A. FIG. 79C is a comparison of the experimental data with the CAFT model predicted flammability envelope. The model agrees very well with the experimental data. Discrepancies may be due to the same factors described in Series 1

VI. Calculation of Flammability Limits of Isoprene in Air at 3 Atmospheres of Pressure

The methods described in Example 13, parts I to IV were also used to calculate the flammability limits of isoprene at an absolute system pressure of 3 atmospheres and 40° C. These results were compared to those of Example 13, parts I to IV at an absolute system pressure of 1 atmosphere and 40° C. This higher pressure was tested because the flammability envelope expands or grows larger as the initial system pressure is increased. The upper flammability limit is affected the most, followed by the limiting oxygen composition. The lower flammability limit is the least affected (see, for example, “Bulletin 627—Flammability Characteristics of Combustible Gases and Vapors” written by Michael G. Zabetakis and published by the former US Bureau of Mines (1965), which is hereby incorporated by reference in its entirety, particular with respect to the calculation of flammability limits).

In FIG. 82, the calculated adiabatic flame temperature is plotted as a function of isoprene (fuel) concentration, expressed in weight percent of the total fuel/nitrogen/oxygen, where the system pressure was initially 3 atmospheres. The calculated flame temperatures are very similar to those determined initially in the 1 atmosphere system (FIG. 83). As a result, when flammability envelopes are generated using the calculated adiabatic flammability data, the curves are very similar (see FIGS. 84 and 85). Therefore, based on these theoretical calculations, a system pressure increase from 1 atmosphere to 3 atmosphere does not result in a significant increase/broadening of the flammability envelope. If desired, these model results may be validated using experimental testing (such as the experimental testing described herein at a pressure of 1 atmosphere).

VII. Summary of Flammability Studies

A calculated adiabatic temperature model was developed for the flammability envelope of the isoprene/oxygen/nitrogen/water/carbon dioxide system at 40° C. and 0 psig. The CAFT model that was developed agreed well with the experimental data generated by the tests conducted in this work. The experimental results from Series 1 and 2 validated the model results from Series A and B.

Example 14 Expression Constructs and Strains

I. Construction of Plasmids Encoding Mevalonate Kinase.

A construct encoding the Methanosarcina mazei lower MVA pathway (Accession numbers NC_(—)003901.1, NC_(—)003901.1, NC_(—)003901.1, and NC_(—)003901.1, which are each hereby incorporated by reference in their entireties) was synthesized with codon optimization for expression in E. coli. This construct is named M. mazei archeal Lower Pathway operon (FIGS. 112A-112C; SEQ ID NO:27) and encodes M. mazei MVK, a putative decarboxylase, IPK, and IDI enzymes. The gene encoding MVK (Accession number NC_(—)003901.1) was PCR amplified using primers MCM165 and MCM177 (Table 11) using the Strategene Herculase II Fusion kit according to the manufacturer's protocol using 30 cycles with an annealing temperature of 55° C. and extension time of 60 seconds. This amplicon was purified using a Qiagen PCR column and then digested at 37° C. in a 10 pt reaction with PmeI (in the presence of NEB buffer 4 and BSA). After one hour, NsiI and Roche buffer H were added for an additional hour at 37° C. The digested DNA was purified over a Qiagen PCR column and ligated to a similarly digested and purified plasmid MCM29 (MCM29 is E. coli TOP10 (Invitrogen) transformed with pTrcKudzu encoding Kudzu isoprene synthase) in an 11 μL reaction 5 μL Roche Quick Ligase buffer 1, 1 μL buffer 2, 1 μL plasmid, 3 μL amplicon, and 1 μL ligase (1 hour at room temperature). MCM 29 is pTrcKudzuKan. The ligation reaction was introduced into Invitrogen TOP10 cells and transformants selected on LA/kan50 plates incubated at 37° C. overnight. The MVK insert in the resulting plasmid MCM382 was sequenced (FIGS. 113A-113C; SEQ ID NO: 28).

TABLE 11 Oligonucleotides. MCM161 M. mazei MVK for CACCATGGTATCCTGTTCTGCG (SEQ ID NO: 120) MCM162 M. mazei MVK rev TTAATCTACTTTCAGACCTTGC (SEQ ID NO: 121) MCM165 M. mazei MVK for w/ gcgaacgATGCATaaaggaggtaaaaaaacATGGTATCCTGTTCTG RBS CGCCGGGTAAGATTTACCTG (SEQ ID NO: 122) MCM177 M. mazei MVK rev Pst gggcccgtttaaactttaactagactTTAATCTACTTTCAGACCTTGC (SEQ ID NO: 123) II. Creation of Strains Overexpressing Mevalonate Kinase and Isoprene Synthase.

Plasmid MCM382 was transformed into MCM331 cells (which contains chromosomal construct gi1.2KKDyI encoding S. cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, and IPP isomerase) that had been grown to midlog in LB medium and washed three times in iced, sterile water. One μL of DNA was added to 50 μL of cell suspension, and this mixture was electroporated in a 2 mm cuvette at 2.5 volts, 25 uFd followed immediately by recovery in 500 μL LB medium for one hour at 37° C. Transformant was selected on LA/kan50 and named MCM391. Plasmid MCM82 was introduced into this strain by the same electroporation protocol followed by selection on LA/kan50/spec50. The resulting strain MCM401 contains a cmp-marked chromosomal construct gi1.2KKDyI, kan-marked plasmid MCM382, and spec-marked plasmid MCM82 (which is pCL PtrcUpperPathway encoding E. faecalis mvaE and mvaS). See Table 12.

TABLE 12 Strains overexpressing mevalonate kinase and isoprene synthase. MCM382 E. coli BL21 (lambdaDE3) pTrcKudzuMVK(M. mazei)GI1.2KKDyI MCM391 MCM331 pTrcKudzuMVK(M. mazei) MCM401 MCM331pTrcKudzuMVK(M. mazei)pCLPtrcUpperpathway MCM396 MCM333pTrcKudzuMVK(M. mazei) MCM406 MCM333pTrcKudzuMVK(M. mazei)pCLPtrcUpperpathway III. Construction of Plasmid MCM376-MVK from M. mazei Archeal Lower in pET200D.

The MVK ORF from the M. mazei archeal Lower Pathway operon (FIGS. 112A-112C; SEQ ID NO:27) was PCR amplified using primers MCM161 and MCM162 (Table 11) using the Invitrogen Platinum HiFi PCR mix. 45 μL of PCR mix was combined with 1 μL template, 1 μL of each primer at 10 μM, and 2 μL water. The reaction was cycled as follows: 94° C. for 2:00; 30 cycles of 94° C. for 0:30, 55° C. for 0:30, and 68° C. for 1:15; and then 72° C. for 7:00, and 4° C. until cool. 3 μL of this PCR reaction was ligated to Invitrogen pET200D plasmid according to the manufacturer's protocol. 3 μL of this ligation was introduced into Invitrogen TOP10 cells, and transformants were selected on LA/kan50. A plasmid from a transformant was isolated and the insert sequenced, resulting in MCM376 (FIGS. 114A-114C; SEQ ID NO:29).

IV. Creation of Expression Strain MCM378.

Plasmid MCM376 was transformed into Invitrogen BL21(DE3) pLysS cells according to the manufacturer's protocol. Transformant MCM378 was selected on LA/kan50.

Example 15 Production of Isoprene by E. coli Expressing the Upper Mevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway (gi1.2KKDyI), Mevalonate Kinase from M. mazei, and Isoprene Synthase from Kudzu and Grown in Fed-Batch Culture at the 20 mL Batch Scale

Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 1 g, and 1000× Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH₂O. The pH was adjusted to 6.8 with ammonium hydroxide (30%) and brought to volume. Media was filter sterilized with a 0.22 micron filter. Glucose (2.5 g) and antibiotics were added after sterilization and pH adjustment.

1000× Trace Metal Solution:

1000× Trace Metal Solution contained citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in di H₂O, pH to 3.0 with HCl/NaOH, then brought to volume and filter sterilized with a 0.22 micron filter.

Strains:

MCM343 cells are BL21 (DE3) E. coli cells containing the upper mevalonic acid (MVA) pathway (pCL Upper), the integrated lower MVA pathway (gi1.2KKDyI), and isoprene synthase from Kudzu (pTrcKudzu).

MCM401 cells are BL21 (DE3) E. coli cells containing the upper mevalonic acid (MVA) pathway (pCL PtrcUpperPathway), the integrated lower MVA pathway (gi1.2KKDyI), and high expression of mevalonate kinase from M. mazei and isoprene synthase from Kudzu (pTrcKudzuMVK(M. mazei)).

Isoprene production was analyzed by growing the strains in 100 mL bioreactors with a 20 mL working volume at a temperature of 30° C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 30° C. A single colony was inoculated into media and grown overnight. The bacteria were diluted into 20 mL of media to reach an optical density of 0.05 measured at 550 nm. The 100 mL bioreactors were sealed, and air was pumped through at a rate of 8 mL/min. Adequate agitation of the media was obtained by stirring at 600 rpm using magnetic stir bars. The off-gas from the bioreactors was analyzed using an on-line Hiden HPR-20 mass spectrometer. Masses corresponding to isoprene, CO₂, and other gasses naturally occurring in air were monitored. Accumulated isoprene and CO₂ production were calculated by summing the concentration (in percent) of the respective gasses over time. Atmospheric CO₂ was subtracted from the total in order to estimate the CO₂ released due to metabolic activity.

Isoprene production from a strain expressing the full mevalonic acid pathway and Kudzu isoprene synthase (MCM343) was compared to a strain that in addition over-expressed MVK from M. mazei and Kudzu isoprene synthase (MCM401) in 100 mL bioreactors. The bacteria were grown under identical conditions in defined media with glucose as carbon source. Induction of isoprene production was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG) to a final concentration of either 100 μM or 200 μM. Off-gas measurements revealed that the strain over-expressing both MVK and isoprene synthase (MCM401) produced significantly more isoprene compared to the strain expressing only the mevalonic acid pathway and Kudzu isoprene synthase (MCM343) as shown in FIGS. 115A-115D. At 100 μM induction, the MCM401 strain produced 2-fold more isoprene compared to the MCM343 strain. At 200 μM IPTG induction, the MCM401 strain produced 3.4-fold more isoprene when compared to the MCM343 strain. Analysis of CO₂ in the off-gas from the bioreactors, which is a measure of metabolic activity, indicates that metabolic activity was independent from IPTG induction and isoprene production.

Example 16 Production of Isoprene by E. coli Expressing the Upper Mevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway (gi1.2KKDyI), Mevalonate Kinase from M. mazei, and Isoprene Synthase from Kudzu and Grown in Fed-Batch Culture at the 15-L Scale

Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in DI H₂O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Modified Trace Metal Solution contained citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in DI H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the upper mevalonic acid (MVA) pathway (pCL PtrcUpperPathway encoding E. faecalis mvaE and mvaS), the integrated lower MVA pathway (gi1.2KKDyI encoding S. cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, and IPP isomerase), and high expression of mevalonate kinase from M. mazei and isoprene synthase from Kudzu (pTrcKudzuMVK (M. mazei)). This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37° C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate 5-L of medium in a 15-L bioreactor.

Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 68 hour fermentation was 3.8 kg. Induction was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The IPTG concentration was brought to 51 μM when the optical density at 550 nm (OD₅₅₀) reached a value of 9. The IPTG concentration was raised to 88 μM when OD₅₅₀ reached 149. Additional IPTG additions raised the concentration to 119 μM at OD₅₅₀=195 and 152 μM at OD₅₅₀=210. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 116. The isoprene level in the off gas from the bioreactor was determined using a Hiden mass spectrometer. The isoprene titer increased over the course of the fermentation to a final value of 23.8 g/L (FIG. 117). The total amount of isoprene produced during the 68 hour fermentation was 227.2 g and the time course of production is shown in FIG. 118. The metabolic activity profile, as measured by TCER, is shown in FIG. 119. The total viable count (total colony forming units) decreased by two orders of magnitude between 10 and 39 hours of fermentation (FIG. 120). The molar yield of utilized carbon that went into producing isoprene during fermentation was 13.0%. The weight percent yield of isoprene from glucose was 6.3%.

Example 17 Production of Isoprene by E. coli Expressing the Upper Mevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway (gi1.2KKDyI), Mevalonate Kinase from M. mazei, and Isoprene Synthase from Kudzu and Grown in Fed-Batch Culture at the 15-L Scale (2×100 μM IPTG induction)

Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in DI H₂O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Modified Trace Metal Solution contained citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in DI H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the upper mevalonic acid (MVA) pathway (pCL PtrcUpperPathway encoding E. faecalis mvaE and mvaS), the integrated lower MVA pathway (gi1.2KKDyI encoding S. cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, and IPP isomerase), and high expression of mevalonate kinase from M. mazei and isoprene synthase from Kudzu (pTrcKudzuMVK(M. mazei)). This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37° C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate 5-L medium in a 15-L bioreactor.

Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 55 hour fermentation was 1.9 kg. Induction was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The IPTG concentration was brought to 111 μM when the optical density at 550 nm (OD₅₅₀) reached a value of 9. The IPTG concentration was raised to 193 μM when OD₅₅₀ reached 155. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 121. The isoprene level in the off gas from the bioreactor was determined using a Hiden mass spectrometer. The isoprene titer increased over the course of the fermentation to a final value of 19.5 g/L (FIG. 122). The total amount of isoprene produced during the 55 hour fermentation was 133.8 g and the time course of production is shown in FIG. 123. Instantaneous volumetric productivity levels reached values as high as 1.5 g isoprene/L broth/hr (FIG. 124). Instantaneous yield levels reached as high as 17.7% w/w (FIG. 125). The metabolic activity profile, as measured by TCER, is shown in FIG. 126. The total viable count (total colony forming units) decreased by two orders of magnitude between 8 and 36 hours of fermentation (FIG. 127). The molar yield of utilized carbon that went into producing isoprene during fermentation was 15.8%. The weight percent yield of isoprene from glucose over the entire fermentation was 7.4%.

In addition, as a control, fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the upper mevalonic acid (MVA) pathway (pCL PtrcUpperPathway encoding E. faecalis mvaE and mvaS), the integrated lower MVA pathway (gi1.2KKDyI encoding S. cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, and IPP isomerase), and high expression of mevalonate kinase from M. mazei and isoprene synthase from Kudzu (pTrcKudzuMVK(M. mazei)). This experiment was carried out to monitor uninduced cell metabolic activity as measured by CER from glucose at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain (MCM401 described above) taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37° C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate 5-L medium in a 15-L bioreactor. Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands.

FIG. 148 compares the CER profiles for the uninduced cells described above and the cells induced by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG) in Examples 16 and 17.

Example 18 Production of Isoprene by E. coli Expressing the Upper Mevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway (gi1.2KKDyI), Mevalonate Kinase from M. mazei, and Isoprene Synthase from Kudzu and Grown in Fed-Batch Culture at the 15-L Scale (1×50 μM IPTG+150 μM IPTG Fed Induction)

Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Modified Trace Metal Solution contained citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in DI H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the upper mevalonic acid (MVA) pathway (pCL PtrcUpperPathway encoding E. faecalis mvaE and mvaS), the integrated lower MVA pathway (gi1.2KKDyI encoding S. cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, and IPP isomerase), and high expression of mevalonate kinase from M. mazei and isoprene synthase from Kudzu (pTrcKudzuMVK(M. mazei)). This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37° C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate 5-L medium in a 15-L bioreactor.

Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 55 hour fermentation was 2.2 kg. Induction was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The IPTG concentration was brought to 51 μM when the optical density at 550 nm (OD₅₅₀) reached a value of 10. In addition to the IPTG spike, at OD₅₅₀=10 a constant feed began and delivered 164 mg of IPTG over 18 hours. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 128. The isoprene level in the off gas from the bioreactor was determined using a Hiden mass spectrometer. The isoprene titer increased over the course of the fermentation to a final value of 22.0 g/L (FIG. 129). The total amount of isoprene produced during the 55 hour fermentation was 170.5 g and the time course of production is shown in FIG. 130. The metabolic activity profile, as measured by TCER, is shown in FIG. 131. When the airflow to the bioreactor was decreased from 8 slpm to 4 slpm for a period of about 1.7 hours, the concentration of isoprene in the offgas increased from 0.51 to 0.92 w/w % (FIG. 132). These elevated levels of isoprene did not appear to have any negative impact on cell metabolic activity as measured by the total carbon dioxide evolution rate (TCER), since TCER declined only 7% between 37.2 and 39.3 hours (FIG. 132). The total viable count (total colony forming units) decreased by two orders of magnitude between 7 and 36 hours of fermentation (FIG. 133). The molar yield of utilized carbon that went into producing isoprene during fermentation was 16.6%. The weight percent yield of isoprene from glucose over the entire fermentation was 7.7%.

Example 19 The Effect of Externally Applied Isoprene on a Wild-Type E. coli Grown in Fed-Batch Culture at the 1-L Scale

Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Modified Trace Metal Solution contained citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in DI H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micron filter.

Fermentation was performed in a 1-L bioreactor with BL21 (DE3) E. coli cells. This experiment was carried out to monitor the effects of isoprene on cell viability and metabolic activity in a glucose fed-batch bioreactor at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain from a frozen vial was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 50 mL was used to inoculate 0.5-L medium in a 1-L bioreactor.

Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was fed to meet metabolic demands. Isoprene was fed into the bioreactor using nitrogen gas as a carrier. The rate of isoprene feeding was 1 g/L/hr during mid-growth phase (OD₅₅₀=31-44) and lasted for a total of 75 minutes (13.2 to 14.4 hours). The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 134. The metabolic activity profile, as measured by TCER, is shown in FIG. 135. The total viable count (total colony forming units) increased by 14-fold during the period when isoprene was introduced into the bioreactor (FIG. 136).

Example 20 Production of Isoprene and Expression of Isoprene Synthase by Saccharomyces cerevisiae

The Kudzu isoprene synthase enzyme was optimized for expression according to a hybrid Saccharomyces cerevisiae/Pichia pastoris codon usage table, synthesized, and cloned into pDONR221:19430 (by DNA 2.0, FIG. 140 for map and FIG. 141 for sequence (SEQ ID NO:38)). A Gateway® Cloning (Invitrogen) reaction was performed according to the manufacturer's protocol: Since pDONR221:19430 was an “entry” vector, the LR Clonase II enzyme (the LR Reaction) was used to introduce the codon-optimized isoprene synthase into the “destination” vector pYES-DEST52 (Invitrogen).

The LR Reaction was then transformed into Top10 chemically competent cells (Invitrogen) according to the manufacturer's protocol, and bacteria harboring pYES-DEST52 plasmids with the isoprene synthase ORF were selected for on LA plates containing 50 μg/ml carbenicillin. Individual positive transformants were tested by colony PCR (see below for primer concentrations and thermocycling parameters) using illustra PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare) with the T7 forward primer and the Yeast isoprene synthase-Rev2 primer (See Table 13).

TABLE 13 Primer sequences for amplifying isoprene synthase. Primer Name Sequence (5′ to 3′) Purpose Yeast HGS - CACCAAAGACTTCATAGACT Forward primer for yeast For2 (SEQ ID NO: 124) optimized isoprene synthase Yeast HGS - AGAGATATCTTCCTGCTGCT Reverse primer for yeast Rev2 (SEQ ID NO: 125) optimized isoprene synthase T7 Forward TAATACGACTCACTATAGGG PCR and sequencing primer (SEQ ID NO: 126)

Plasmids that yielded a PCR fragment of the correct size (1354 bp) were purified by miniprep (Qiagen) and sent for sequencing (Quintara Biosciences, Berkeley, Calif.) with the T7 Forward and Yeast isoprene synthase-For2 primers (See Table 13). Results from sequencing runs were compared to the known sequence of pDONR221:19430 (using Vector NTI software, Invitrogen), and a single plasmid, pDW14, was selected for further study (FIG. 142A for map and FIGS. 142B and C for the complete sequence (SEQ ID NO:39)). The sequence of pDW14 diverged from that of pDONR221:19430 by a single nucleotide (marked in bold in FIG. 142B). The single nucleotide change (G to A) did not result in a change in the ORF, since it was in the third position of a lysine-encoding codon.

Purified pDW14 was transformed into Saccharomyces cerevisiae strain INVSc-1 using the protocol described in the S. c. EasyComp Transformation kit (Invitrogen). INVSc-1 strains harboring pDW14 or pYES-DEST52 (which contains an intact URA3 gene) were selected for and maintained on SC Minimal Medium with 2% glucose without uracil, as described in the pYES-DEST52 Gateway Vector manual (Invitrogen). Two independent isolates of INVSc-1 containing pDW14 and a single control strain with pYES-DEST52 were chosen for further analysis.

To induce isoprene synthase expression, cultures were grown overnight in liquid SC Minimal Medium. The cultures were then diluted to an OD₆₀₀ of approximately 0.2 and grown for 2-3 hours. Cultures were spun by centrifugation, washed once, resuspended in an equal volume (10 ml) of SC minimal medium with 1% raffinose, 2% galactose without uracil, and grown overnight to induce the expression of isoprene synthase. The OD₆₀₀ of the strains was determined (FIG. 144A), and strains were harvested by centrifugation and resuspended in 2 ml of lysis buffer (a 1:1 mix of 50% glycerol and PEB pH 7.4: Tris Base 2.423 g/L, MgCl₂ (Anhydrous) 1.904 g/L, KCl 14.910 g/L, DTT 0.154 g/L, Glycerol 50 mL/L).

The lysis mixtures were passed through a french press three times, and lysates were analyzed by SDS-PAGE. For Coomassie gel analysis (FIG. 143A), samples were diluted 1:1 with 2×SDS loading buffer with reducing agent, loaded (20 μl total volume) onto a 4-12% bis-tris gel, run in MES buffer, and stained using SimplyBlue SafeStain according to the manufacturer's protocol (the Invitrogen Novex system).

The WesternBreeze kit (Invitrogen) was used for transfer and chromogenic detection of isoprene synthase on a nitrocellulose membrane. The primary antibody was 1799A 10 week diluted 1:1000 in Invitrogen antibody diluent. Primary antibody binding was followed by development with a secondary antibody labeled with Alexa Fluor 488 (Invitrogen Catalog No. A-11008) to permit quantitative signal determination. The western blot procedure was carried out as described by Invitrogen. The fluorescence signal was recorded with a Molecular Dynamics Storm instrument using the blue filter setting and quantitatively analyzed with the Molecular Dynamics ImageQuant image analysis software package. Specific activity of the library members was calculated from the ratio of the amount of isoprene produced divided by either the A600 of the induction cultures or the isoprene synthase protein concentration determined by western blot. FIG. 143B shows that isoprene synthase was present in the induced INVSc-1 strains harboring pDW14 (lanes 2 and 3) in comparison to the control harboring pYES-DEST52 (lane 1).

The DMAPP assay for isoprene synthase headspace was performed on 25 μL of the lysate from each strain for which 5 μL 1 M MgCl2, 5 μL 100 mM DMAPP, and 65 μL 50 mM Tris pH 8 were added. The reaction was performed at 30° C. for 15 minutes in a gas tight 1.8 mL GC tube. Reactions were terminated by addition of 100 μL 250 mM EDTA pH 8. FIG. 144B showed the specific activity values (in μg HG/L/OD) of the induced strains harboring pDW14 in comparison to the control. Induced strains harboring pDW14 displayed approximately 20× higher activity than the control lacking isoprene synthase.

PCR Cycling Parameters

Illustra PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare) were used with oligonucleotide primer pairs at a concentration of 0.4 μM each in 25 μl total volume/reaction. For analysis of plasmids resulting from the LR Clonase reaction (Invitrogen), a small amount of bacteria from individual colonies on a selective plate was added to each tube containing the PCR mix described above. The reaction cycle was as follows: 1) 95° C. for 4 minutes; 2) 95° C. for 20 seconds; 3) 52° C. for 20 seconds; 4) 72° C. for 30 seconds; 5 cycles of steps 2 through 4; 5) 95° C. for 20 seconds; 6) 55° C. for 20 seconds; 7) 72° C. for 30 seconds; 25 cycles of steps 5 through 7, 72° C. for 10 minutes, and 4° C. until cool.

Example 21 Production of Isoprene in Pseudomonas and Other Gram Negative Bacteria Construction of pBBRSHGSOpt2_(—)2, Conjugation in Pseudomonas and Measurement of Isoprene Synthase Activity

A gene encoding isoprene synthase from Pueraria lobata (Kudzu plant) was codon-optimized for different microbial species of interest (Table 14; fluo-opt2v2 was the sequence chosen) and was synthesized by DNA2.0, Menlo Park, Calif. The map and sequence of fluo-opt2v2 can be found in FIGS. 145A and 145B (SEQ ID NO:40). HindIII and BamHI restriction sites were added to the synthesized sequence for easier cloning, and a RBS was added in front of the ATG to enhance transcription.

Number of rare codons, as a function of the microbial species, in different versions of codon-optimized isoprene synthase from Pueraria lobata. Several rounds of optimization led to a gene with no rare codons in the all the species of interest.

TABLE 14 Number of rare codons. fluo-opt1 Organism (quote) fluo-opt2 fluo-opt3 E. coli opt fluo-opt2v2 Pseudomonas fluorescens Pf-5 19 X X 57 0 Phodopseudomonas palustris 37 13 3 74 0 CGA009 Pseudomonas putida F1  0 0 0 29 0 Corynebacterium glutamicum 4 (Ser) 0 0 0 0 (ATCC) Pseudomonas fluorescens PfO-1 1 (Val) 0 0 57 0

The gene was provided by DNA2.0 in a cloning vector. The vector was digested with HindIII/BamHI, the band corresponding to the insert of interest was gel-purified, and relegated with HindIII/BamHI-digested pBBR1MCS5 (Kovach et al, Gene 166:175-176, 1995, which is incorporated by reference in its entirety, particularly with respect to pBBR1MCS5), FIG. 146A for map and FIGS. 146B and C for sequence (SEQ ID NO:41). This resulted in plasmid pBBR5HGSOpt2_(—)2 (FIG. 147A for map and FIGS. 147B and C for sequence (SEQ ID NO:42)) in which isoprene synthase was expressed from the lac promoter presented in pBBR1MCS5.

The vector was transformed in E. coli 517-1 and mated with Pseudomonas putida F1 ATCC700007 and Pseudomonas fluorescens ATCC 13525. After conjugation on LB, selection for plasmid-harboring Pseudomonas strains was on M9+16 mM sodium citrate+Gentamicin 50 μg/ml. Presence of the plasmid in the strains thus generated was checked by plasmid preparation using the Qiagen kit (Valencia, Calif.).

Isoprene synthase activities of the recombinant strains P. putida, pBBR5HGSOpt2_(—)2 and P. fluorescens, pBBR5HGSOpt2_(—)2 were assayed by growing the strains in TM3 medium (as described in Example 1 Part II)+10 g/L glucose, harvesting the biomass in mid-log phase, breaking the cells by French Press and proceeding with the DMAPP assay. Results of the assay were presented in Table 15. The presence of activity measured by the DMAPP assay confirmed that isoprene synthase was expressed in Pseudomonas.

Isoprene synthase activity was examined in Pseudomonas putida and Pseudomonas fluorescens expressing isoprene synthase from the lac promoter, using plasmid pBBR5HGSOpt2_(—)2

TABLE 15 Isoprene synthase activity in Pseudomonas putida and Pseudomonas fluorescens. Isoprene synthase activity Strain OD mg isoprene/(L · h · OD) P. fluorescens, pBBR5HGSOpt2_2 1.46 0.96 P. putida, pBBR5HGSOpt2_2 3.44 0.65 Control (P. putida w/o plasmid) 8.32 To be determined

Example 22 Growth of E. coli and Pseudomonas Strains on Sugar Cane Compared to Glucose, and Expression of Isoprene Synthase Using Both Substrates

I. Preparation of Liquid Sugar Cane

Crystallized raw cane sugar was dissolved in water in the following way: 750 g H₂O was added to 250 g sugar. The solution was stirred and gently heated until dissolution. Some material was not soluble. The weight of the solution was adjusted to 1 kg after dissolution to replenish the evaporated water. The volume of the solution was measured to be 940 mL. Hence the concentration of the solution was 265 g/L. The product label claimed 14 g of carbohydrate for 15 g of raw sugar cane. Hence the carbohydrate concentration of the solution was 248 g/L. Dry solids were measured to be 24.03%, close enough of the expected 250 g/kg. pH of the solution was 5.49. Glucose concentration was measured using an enzymatic/spectrophotometric assay, with glucose oxidase. The glucose concentration was 17.4 g/L.

As a majority of microorganisms do not use sucrose, but can use glucose and fructose, the solution was split in two. One half was autoclaved once for 30 minutes (sugar cane as is). Some inversion resulted, as the glucose content increased to 29.75 g/L (See FIG. 149). The other half of the solution was adjusted to pH 4.0 using phosphoric acid, then the solution was inverted by autoclaving (inverted sugar cane). Three cycles of 30 min were sufficient to obtain complete inversion, as shown on FIG. 149. Both solutions were used for the growth curves described below.

II. Growth Curves of Different Strains of E. coli and Pseudomonas on Sugar Cane Compared to Glucose

One colony of each of the strains presented in Table 16 was inoculated in 25 ml TM3+10 g/L glucose, and was grown overnight at 30° C. and 200 rpm. TM3 is described in Example 7, Section II. The morning after, 1 ml of each culture was used to inoculate flasks containing 25 mL TM3 and 10 g/L glucose, 10 g/L sugar cane as is, or 10 g/L inverted sugar cane (sugar cane solutions described above). The flasks were incubated at 30° C. and 200 rpm and samples were taken regularly to measure OD600. FIGS. 150 and 151 show that growth rate and biomass yield were comparable for glucose and inverted sugar cane, both for Pseudomonas and E. coli strains. P. fluorescens showed some signs of being able to use sugar cane which has not been inverted too.

TABLE 16 Strains used in this study. Strain Escherichia coli BL21 MG1655 ATCC11303 B REL 606 Pseudomonas putida F1 (ATCC700007) Fluorescens (ATCC13525) III. Comparison of Isoprene Production from E. coli Expressing Isoprene Synthase when Grown on Glucose or Sugar Cane

E. coli MCM401 (BL21(DE3)) containing the full MVA pathway, mevalonate kinase from M. mazei and isoprene synthase from Pueraria lobata, as described in Example 14, Section II was grown in TM3+either 10 g/L glucose or 10 g/L inverted sugar cane (based on carbohydrate concentration of the syrup). Flasks were inoculated from an overnight culture on TM3+10 g/L glucose at an OD₆₀₀=0.2. Antibiotics were added where needed. After two hours, the E. coli cultures were induced with 400 μM IPTG. After 6 hours of growth, isoprene production and isoprene synthase activities, using the DMAPP assay as described in Example 2B, were measured. Results are presented in Table 17 and illustrate clearly that inverted sugar cane is equivalent to glucose in terms of isoprene and isoprene synthase production on a per cell basis.

TABLE 17 Isoprene Isoprene synthase activity production mg mg isoprene/ Strain Carbon Source OD isoprene/(L · h · OD) (L · h · OD) MCM401 Glucose 2.20 21.06 8.98 MCM401 Sugar cane 2.32 20.20 9.23 inverted

Example 23 Construction of E. coli Strains Expressing the S. cerevisiae gi1.2KKDyI Operon, P. alba Isoprene Synthase, M. mazei Mevalonate Kinase, pCL Upper MVA (E. faecalis mvaE and mvaS) and ybhE (pgl)

(i) Construction of Strain EWL201 (BL21, Cm-GI1.2-KKDyI)

E. coli BL21 (Novagen brand, EMD Biosciences, Inc.) was a recipient strain, transduced with MCM331 P1 lysate (lysate prepared according to the method described in Ausubel, et al., Current Protocols in Molecular Biology. John Wiley and Sons, Inc.). MCM331 cells contain chromosomal construct gi1.2KKDyI encoding S. cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, and IPP isomerase (i.e., the gi1.2-KKDyI operon from S. cerevisiae). Transductants were selected for by spreading cells onto L Agar and 20 μg/μl chloramphenicol. The plates were incubated overnight at 30° C. Analysis of transductants showed no colonies on control plates (water+cells control plate for reversion and water and P1 lysate control plate for lysate contamination.

Four transductants were picked and used to inoculate 5 mL L Broth and 20 μg/μl chloramphenicol. The cultures were grown overnight at 30° C. with shaking at 200 rpm. To make genomic DNA preps of each transductant for PCR analysis, 1.5 mL of overnight cell culture were centrifuged. The cell pellet was resuspended with 400 μl Resuspension Buffer (20 mM Tris, 1 mM EDTA, 50 mM NaCl, pH 7.5) and 4 μl RNase, DNase-free (Roche) was added. The tubes were incubated at 37° C. for 30 minutes followed by the addition of 4 μl 10% SDS and 4 μl of 10 mg/ml Proteinase K stock solution (Sigma-Aldrich). The tubes were incubated at 37° C. for 1 hour. The cell lysate was transferred into 2 ml Phase Lock Light Gel tubes (Eppendorf) and 200 μl each of saturated phenol pH7.9 (Ambion Inc.) and chloroform were added. The tubes were mixed well and microcentrifuged for 5 minutes. A second extraction was done with 400 μl chloroform and the aqueous layer was transferred to a new eppendorf tube. The genomic DNA was precipitated by the addition of 1 ml of 100% ethanol and centrifugation for 5 minutes. The genomic DNA pellet was washed with 1 ml 70% ethanol. The ethanol was removed and the genomic DNA pellet was allowed to air dry briefly. The genomic DNA pellet was resuspended with 200 μl TE.

Using Pfu Ultra II DNA polymerase (Stratagene) and 200 ng/μl of genomic DNA as template, 2 different sets of PCR reaction tubes were prepared according to manufacturer's protocol. For set 1, primers MCM130 and GB Cm-Rev (Table 18) were used to ensure transductants were successfully integrated into the attTn7 locus. PCR parameters for set 1 were 95° C. for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C. for 25 seconds, 72° C. for 25 seconds (repeat steps 2-4 for 28 cycles), 72° C. for 1 minute. For set 2, primers MVD For and MVD Rev (Table 18) were used to ensure that the gi1.2-KKDyI operon integrated properly. PCR parameters for set 2 were 95° C. for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C. for 25 seconds, 72° C. for 10 seconds (repeat steps 2-4 for 28 cycles), 72° C. for 1 minute. Analysis of PCR amplicons on a 1.2% E-gel (Invitrogen Corp.) showed that all 4 transductant clones were correct. One was picked and designated as strain EWL201.

(ii) Construction of Strain EWL204 (BL21, Loopout-GI1.2-KKDyI)

The chloramphenicol marker was looped out of strain EWL201 using plasmid pCP20 as described by Datsenko and Wanner (2000) (Datsenko et al., Proc Natl. Acad. Sci. USA 97:6640-6645, 2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. (Datsenko et al., PNAS, 97: 6640-6645, 2000). EWL201 cells were grown in L Broth to midlog phase and then washed three times in ice-cold, sterile water. An aliquot of 50 μl of cell suspension was mixed with 1 μl of pCP20 and the cell suspension mixture was electroporated in a 2 mm cuvette (Invitrogen Corp.) at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator (Bio-Rad Inc.). 1 ml of LB was immediately added to the cells, then transferred to a 14 ml polypropylene tube (Sarstedt) with a metal cap. Cells were allowed to recover by growing for 1 hour at 30° C. Transformants were selected on L Agar and 20 μg/μl chloramphenicol and 50 μg/μl carbenicillin and incubated at 30° C. overnight. The next day, a single clone was grown in 10 ml L Broth and 50 μg/μl carbenicillin at 30° C. until early log phase. The temperature of the growing culture was then shifted to 42° C. for 2 hours. Serial dilutions were made, the cells were then spread onto LA plates (no antibiotic selection), and incubated overnight at 30° C. The next day, 20 colonies were picked and patched onto L Agar (no antibiotics) and LA and 20 μg/μl chloramphenicol plates. Plates were then incubated overnight at 30° C. Cells able to grow on LA plates, but not LA and 20 μg/μl chloramphenicol plates, were deemed to have the chloramphenicol marker looped out (picked one and designated as strain EWL204).

(iii) Construction of Plasmid pEWL230 (pTrc P. alba)

Generation of a synthetic gene encoding Populus alba isoprene synthase (P. alba HGS) was outsourced to DNA2.0 Inc. (Menlo Park, Calif.) based on their codon optimization method for E. coli expression. The synthetic gene was custom cloned into plasmid pET24a (Novagen brand, EMD Biosciences, Inc.) and delivered lyophilized (FIGS. 152, 153A-B; SEQ ID NO:43).

A PCR reaction was performed to amplify the P. alba isoprene synthase (P. alba HGS) gene using pET24 P. alba HGS as the template, primers MCM182 and MCM192, and Herculase II Fusion DNA polymerase (Stratagene) according to manufacturer's protocol. PCR conditions were as follows: 95° C. for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C. for 20 seconds, 72° C. for 1 minute, repeat for 25 cycles, with final extension at 72° C. for 3 minutes. The P. alba isoprene synthase PCR product was purified using QIAquick PCR Purification Kit (Qiagen Inc.).

P. alba isoprene synthase PCR product was then digested in a 20 μl reaction containing 1 μl BspHI endonuclease (New England Biolabs) with 2 μl 10×NEB Buffer 4. The reaction was incubated for 2 hours at 37° C. The digested PCR fragment was then purified using the QIAquick PCR Purification Kit. A secondary restriction digest was performed in a 20 μl reaction containing 1 μl PstI endonuclease (Roche) with 4,110× Buffer H. The reaction was incubated for 2 hours at 37° C. The digested PCR fragment was then purified using the QIAquick PCR Purification Kit. Plasmid pTrcHis2B (Invitrogen Corp.) was digested in a 20 μl reaction containing 1 μl NcoI endonuclease (Roche), 1 μl PstI endonuclease, and 2 μl 10× Buffer H. The reaction was incubated for 2 hours at 37° C. The digested pTrcHis2B vector was gel purified using a 1.2% E-gel (Invitrogen Corp.) and extracted using the QIAquick Gel Extraction Kit (Qiagen) (FIG. 154). Using the compatible cohesive ends of BspHI and NcoI sites, a 20 μl ligation reaction was prepared containing 5 μl P. alba isoprene synthase insert, 2 μl pTrc vector, 1 μl T4 DNA ligase (New England Biolabs), 2 μl 10× ligase buffer, and 10 μl ddH₂O. The ligation mixture was incubated at room temperature for 40 minutes. The ligation mixture was desalted by floating a 0.025 μm nitrocellulose membrane filter (Millipore) in a petri dish of ddH₂O and applying the ligation mixture gently on top of the nitrocellulose membrane filter for 30 minutes at room temperature. MCM446 cells (see Section II) were grown in LB to midlog phase and then washed three times in ice-cold, sterile water. An aliquot of 50 μl of cell suspension was mixed with 5 μl of desalted pTrc P. alba HGS ligation mix. The cell suspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator. 1 ml of LB is immediately added to the cells, then transferred to a 14 ml polypropylene tube (Sarstedt) with a metal cap. Cells were allowed to recover by growing for 2 hour at 30° C. Transformants were selected on L Agar and 50 μg/μl carbenicillin and 10 mM mevalonic acid and incubated at 30° C. The next day, 6 transformants were picked and grown in 5 ml L Broth and 50 μg/μl carbenicillin tubes overnight at 30° C. Plasmid preps were performed on the overnight cultures using QIAquick Spin Miniprep Kit (Qiagen). Due to the use of BL21 cells for propagating plasmids, a modification of washing the spin columns with PB Buffer 5× and PE Buffer 3× was incorporated to the standard manufacturer's protocol for achieving high quality plasmid DNA. Plasmids were digested with PstI in a 20 μl reaction to ensure the correct sized linear fragment. All 6 plasmids were the correct size and shipped to Quintara Biosciences (Berkeley, Calif.) for sequencing with primers MCM65, MCM66, EL1000 (Table 18). DNA sequencing results showed all 6 plasmids were correct. One plasmid was picked designated as plasmid EWL230 (FIGS. 155, 156A-B; SEQ ID NO:44).

iv) Construction of Plasmid pEWL244 (pTrc P. alba-mMVK)

A PCR reaction was performed to amplify the Methanosarcina mazei (M. mazei) MVK gene using MCM376 as the template (see section (v) below), primers MCM165 and MCM177 (see Table 18), and Pfu Ultra II Fusion DNA polymerase (Stratagene) according to manufacturer's protocol. PCR conditions were as follows: 95° C. for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C. for 25 seconds, 72° C. for 18 seconds, repeat for 28 cycles, with final extension at 72° C. for 1 minute. The M. mazei MVK PCR product was purified using QIAquick PCR Purification Kit (Qiagen Inc.).

The M. mazei MVK PCR product was then digested in a 40 μl reaction containing 8 μl PCR product, 2 μl PmeI endonuclease (New England Biolabs), 4 μl 10×NEB Buffer 4, 4 μl 10×NEB BSA, and 22 μl of ddH₂O. The reaction was incubated for 3 hours at 37° C. The digested PCR fragment was then purified using the QIAquick PCR Purification Kit. A secondary restriction digest was performed in a 47 μl reaction containing 2 μl NsiI endonuclease (Roche), 4.7 μl 10× Buffer H, and 40 μl of PmeI digested M. mazei MVK fragment. The reaction was incubated for 3 hours at 37° C. The digested PCR fragment was then gel purified using a 1.2% E-gel and extracted using the QIAquick Gel Extraction Kit. Plasmid EWL230 was digested in a 40 μl reaction containing 10 μl plasmid, 2 μl PmeI endonuclease, 4 μl 10×NEB Buffer 4, 4 μl 10×NEB BSA, and 20 μl of ddH₂O. The reaction was incubated for 3 hours at 37° C. The digested PCR fragment was then purified using the QIAquick PCR Purification Kit. A secondary restriction digest was performed in a 47 μl reaction containing 2 μl PstI endonuclease, 4.7 μl 10× Buffer H, and 40 μl of PmeI digested EWL230 linear fragment. The reaction was incubated for 3 hours at 37° C. The digested PCR fragment was then gel purified using a 1.2% E-gel and extracted using the QIAquick Gel Extraction Kit (FIG. 157). Using the compatible cohesive ends of NsiI and PstI sites, a 20 μl ligation reaction was prepared containing 8 μl M. mazei MVK insert, 3 μl EWL230 plasmid, 1 μl T4 DNA ligase, 2 μl 10× ligase buffer, and 6 μl ddH₂O. The ligation mixture was incubated overnight at 16° C. The next day, the ligation mixture was desalted by floating a 0.025 μm nitrocellulose membrane filter in a petri dish of ddH₂O and applying the ligation mixture gently on top of the nitrocellulose membrane filter for 30 minutes at room temperature. MCM446 cells were grown in LB to midlog phase and then washed three times in ice-cold, sterile water. An aliquot of 50 μl of cell suspension was mixed with 5 μl of desalted pTrc P. alba-mMVK ligation mix. The cell suspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator. 1 ml of LB is immediately added to the cells, then the cells are transferred to a 14 ml polypropylene tube with a metal cap. Cells were allowed to recover by growing for 2 hour at 30° C. Transformants were selected on LA and 50 μg/μl carbenicillin and 5 mM mevalonic acid plates and incubated at 30° C. The next day, 6 transformants were picked and grown in 5 ml LB and 50 μg/μl carbenicillin tubes overnight at 30° C. Plasmid preps were performed on the overnight cultures using QIAquick Spin Miniprep Kit. Due to the use of BL21 cells for propagating plasmids, a modification of washing the spin columns with PB Buffer 5× and PE Buffer 3× was incorporated to the standard manufacturer's protocol for achieving high quality plasmid DNA. Plasmids were digested with PstI in a 20 μl reaction to ensure the correct sized linear fragment. Three of the 6 plasmids were the correct size and shipped to Quintara Biosciences for sequencing with primers MCM65, MCM66, EL1000, EL1003, and EL1006 (Table 18). DNA sequencing results showed all 3 plasmids were correct. One was picked and designated as plasmid EWL244 (FIGS. 158 and 159A-B; SEQ ID NO:45).

v) Construction of Plasmid MCM376-MVK from M. mazei Archaeal Lower in pET200D.

The MVK ORF from the M. mazei archaeal Lower Pathway operon (FIGS. 160A-C; SEQ ID NO:46) was PCR amplified using primers MCM161 and MCM162 (Table 18) using the Invitrogen Platinum HiFi PCR mix. 45 μL of PCR mix was combined with 1 μL template, 1 μL of each primer at 10 μM, and 2 μL water. The reaction was cycled as follows: 94° C. for 2:00 minutes; 30 cycles of 94° C. for 0:30 minutes, 55° C. for 0:30 minutes and 68° C. for 1:15 minutes; and then 72° C. for 7:00 minutes, and 4° C. until cool. 3 μL of this PCR reaction was ligated to Invitrogen pET200D plasmid according to the manufacturer's protocol. 3 μL of this ligation was introduced into Invitrogen TOP10 cells, and transformants were selected on LA/kan50. A plasmid from a transformant was isolated and the insert sequenced, resulting in MCM376 (FIGS. 161A-C).

vi) Construction of Strain EWL251 (BL21(DE3), Cm-GI1.2-KKDyI, pTrc P. alba-mMVK)

MCM331 cells (which contain chromosomal construct gi1.2KKDyI encoding S. cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, and IPP isomerase) were grown in LB to midlog phase and then washed three times in ice-cold, sterile water. Mixed 50 μl of cell suspension with 1 μl of plasmid EWL244. The cell suspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator. 1 ml of LB is immediately added to the cells, and then the cells were transferred to a 14 ml polypropylene tube with a metal cap. Cells were allowed to recover by growing for 2 hours at 30° C. Transformants were selected on LA and 50 μg/μl carbenicillin and 5 mM mevalonic acid plates and incubated at 37° C. One colony was selected and designated as strain EWL251.

vii) Construction of Strain EWL256 (BL21(DE3), Cm-GI1.2-KKDyI, pTrc P. alba-mMVK, pCL Upper MVA)

EWL251 cells were grown in LB to midlog phase and then washed three times in ice-cold, sterile water. Mixed 50 μl of cell suspension with 1 μl of plasmid MCM82 (comprising pCL PtrcUpperPathway (also known as “pCL Upper MVA”), encoding E. faecalis mvaE and mvaS). Plasmid pCL Ptrc Upper Pathway was constructed as described in Example 8 above. The cell suspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and 25 gd using a Gene Pulser Electroporator. 1 ml of LB was immediately added to the cells. Cells were then transferred to a 14 ml polypropylene tube with a metal cap. Cells were allowed to recover by growing for 2 hours at 30° C. Transformants were selected on LA and 50 μg/μl carbenicillin and 50 μg/μl spectinomycin plates and incubated at 37° C. One colony was picked and designated as strain EWL256.

TABLE 18 Primer Sequences Primer name Primer sequence MCM130 ACCAATTGCACCCGGCAGA (SEQ ID NO: 127) GB Cm GCTAAAGCGCATGCTCCAGAC (SEQ ID NO: 128) Rev MVD GACTGGCCTCAGATGAAAGC (SEQ ID NO: 129) For MVD CAAACATGTGGCATGGAAAG (SEQ ID NO: 130) Rev MCM182 GGGCCCGTTTAAACTTTAACTAGACTCTGCAGTTAGCGTTCAAACGGCAGAA (SEQ ID NO: 131) MCM192 CGCATGCATGTCATGAGATGTAGCGTGTCCACCGAAAA (SEQ ID NO: 132) MCM65 ACAATTTCACACAGGAAACAGC (SEQ ID NO: 133) MCM66 CCAGGCAAATTCTGTTTTATCAG (SEQ ID NO: 106) EL1000 GCACTGTCTTTCCGTCTGCTGC (SEQ ID NO: 134) MCM165 GCGAACGATGCATAAAGGAGGTAAAAAAACATGGTATCCTGTTCTGCGCCGGG TAAGATTTACCTG (SEQ ID NO: 122) MCM177 GGGCCCGTTTAAACTTTAACTAGACTTTAATCTACTTTCAGACCTTGC (SEQ ID NO: 123) EL1003 GATAGTAACGGCTGCGCTGCTACC (SEQ ID NO: 137) EL1006 GACAGCTTATCATCGACTGCACG (SEQ ID NO: 138) MCM161 CACCATGGTATCCTGTTCTGCG (SEQ ID NO: 120) MCM162 TTAATCTACTTTCAGACCTTGC (SEQ ID NO: 121) viii) Construction of Strain RM111608-2 (Cm-GI1.2-KKDyI, pTrc P. alba-mMVK, pCL Upper MVA, pBBRCMPGI1.5-pgl)

The BL21 strain of E. coli producing isoprene (EWL256) was constructed with constitutive expression of the ybhE gene (encoding E. coli 6-phosphogluconolactonase) on a replicating plasmid pBBR1MCS5 (Gentamycin) (obtained from Dr. K. Peterson, Louisiana State University).

FRT-based recombination cassettes, and plasmids for Red/ET-mediated integration and antibiotic marker loopout were obtained from Gene Bridges GmbH (Germany). Procedures using these materials were carried out according to Gene Bridges protocols. Primers Pgl-F (SEQ ID NO:139) and PglGI1.5-R (SEQ ID NO:140) were used to amplify the resistance cassette from the FRT-gb2-Cm-FRT template using Stratagene Herculase II Fusion kit according to the manufacturer's protocol. The PCR reaction (50 μl, final volume) contained: 5 μL buffer, 1 μL template DNA (FRT-gb2-Cm-F from Gene Bridges), 10 pmols of each primer, and 1.5 μL 25 mM dNTP mix, made to 50 μL with dH₂O. The reaction was cycled as follows: 1×2 minutes, 95° C. then 30 cycles of (30 seconds at 95° C.; 30 seconds at 63° C.; 3 minutes at 72° C.).

The resulting PCR product was purified using the QiaQick PCR purification kit (Qiagen) and electroporated into electrocompetent MG1655 cells harboring the pRed-ET recombinase-containing plasmid as follows. Cells were prepared by growing in 5 mLs of L broth to and OD600˜0.6 at 30° C. The cells were induced for recombinase expression by the addition of 4% arabinose and allowed to grow for 30 minutes at 30° C. followed by 30 minutes of growth at 37° C. An aliquot of 1.5 mLs of the cells was washed 3-4 times in ice cold dH₂O. The final cell pellet was resuspended in 40 μL of ice cold dH₂O and 2-5 μL of the PCR product was added. The electroporation was carried out in 1-mm gap cuvettes, at 1.3 kV in a Gene Pulser Electroporator (Bio-Rad Inc.). Cells were recovered for 1-2 hours at 30° C. and plated on L agar containing chloramphenicol (5 μg/mL). Five transformants were analyzed by PCR and sequencing using primers flanking the integration site (2 primer sets: pgl and 49 rev and 3′ EcoRV-pglstop; Bottom Pgb2 and Top GB's CMP (946)). A correct transformant was selected and this strain was designated MG1655 GI1.5-pgl::CMP.

The chromosomal DNA of MG1655 GI1.5-pgl::CMP was used as template to generate a PCR fragment containing the FRT-CMP-FRT-GI1.5-ybhE construct. This construct was cloned into pBBR1MCS5 (Gentamycin) as follows. The fragment, here on referred to as CMP-GI1.5-pgl, was amplified using the 5′ primer Pglconfirm-F (SEQ ID NO:141) and 3′ primer 3′ EcoRV-pglstop (SEQ ID NO:142). The resulting fragment was cloned using the Invitrogen TOPO-Blunt cloning kit into the plasmid vector pCR-Blunt II-TOPO as suggested from the manufacturer. The NsiI fragment harboring the CMP-GI1.5-pgl fragment was cloned into the PstI site of pBBR1MCS5 (Gentamycin). A 20 μl ligation reaction was prepared containing 5 μl CMP-GI1.5-pgl insert, 2 μl pBBR1MCS5 (Gentamycin) vector, 1 μl T4 DNA ligase (New England Biolabs), 2 μl 10× ligase buffer, and 10 μl ddH₂O. The ligation mixture was incubated at room temperature for 40 minutes then 2-4 μL were electroporated into electrocompetent Top10 cells (Invitrogen) using the parameters disclosed above. Transformants were selected on L agar containing 10 μg/ml chloramphenicol and 5 μg/ml Gentamycin. The sequence of the selected clone was determined using a number of the primers described above as well as with the in-house T3 and Reverse primers provided by Sequetech, CA. This plasmid was designated pBBRCMPGI1.5-pgl (FIGS. 162, 163A-B and SEQ ID NO:48).

Plasmid pBBRCMPGI1.5-pgl was electroporated into EWL256, as described herein and transformants were plated on L agar containing Chloramphenicol (10 μg/mL), Gentamycin (5 lag/mL), spectinomycin (50 μg/mL), and carbenicillin (50 μg/mL). One transformant was selected and designated strain RM111608-2.

Primers:

Pgl-F (SEQ ID NO: 139) 5′-ACCGCCAAAAGCGACTAATTTTAGCTGTTACAGTCAGTTGAATTAAC CCTCACTAAAGGGCGGCCGC-3′ PglGI1.5-R (SEQ ID NO: 140) 5′-GCTGGCGATATAAACTGTTTGCTTCATGAATGCTCCTTTGGGTTACC TCCGGGAAACGCGGTTGATTTGTTTAGTGGTTGAATTATTTGCTCAGGAT GTGGCATAGTCAAGGGCGTGACGGCTCGCTAATACGACTCACTATAGGG CTCGAG-3′ 3′ EcoRV-pglstop: (SEQ ID NO: 142) 5′-CTT GAT ATC TTA GTG TGC GTT AAC CAC CAC (SEQ ID NO: 136) pgl +49 rev: CGTGAATTTGCTGGCTCTCAG (SEQ ID NO: 135) Bottom Pgb2: GGTTTAGTTCCTCACCTTGTC (SEQ ID NO: 92) Top GB's CMP (946): ACTGAAACGTTTTCATCGCTC Pglconfirm-F (SEQ ID NO: 141) 5′-ACCGCCAAAAGCGACTAATTTTAGCT-3′

Example 24 Improvement of Isoprene Production by Constitutive Expression of ybhE (pgl) in E. coli

This example shows production of isoprene in a strain constitutively expressing E. coli ybhE (pgl) compared to a control strain expressing ybhE at wild-type levels (i.e., EWL256). The gene ybhE (pgl) encodes E. coli 6-phosphogluconolactonase that suppresses posttranslational gluconylation of heterologously expressed proteins and improves product solubility and yield while also improving biomass yield and flux through the pentose phosphate pathway (Aon et al., Applied and Environmental Microbiology, 74(4): 950-958, 2008).

i) Small Scale Analysis

Media Recipe (per liter fermentation media): K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 1 g, 1000× Trace Metals Solution 1 ml. All of the components were added together and dissolved in diH₂O. The pH was adjusted to 6.8 with ammonium hydroxide (30%) and brought to volume. Media was filter-sterilized with a 0.22 micron filter. Glucose 5.0 g and antibiotics were added after sterilization and pH adjustment.

1000× Trace Metal Solution (per liter fermentation media): Citric Acid*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄.7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in diH₂O. The pH is adjusted to 3.0 with HCl/NaOH, and then the solution is brought to volume and filter-sterilized with a 0.22 micron filter.

(a) Experimental Procedure

Isoprene production was analyzed by growing the strains in a Cellerator™ from MicroReactor Technologies, Inc. The working volume in each of the 24 wells was 4.5 mL. The temperature was maintained at 30° C., the pH setpoint was 7.0, the oxygen flow setpoint was 20 sccm and the agitation rate was 800 rpm. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 30° C. A single colony was inoculated into media with antibiotics and grown overnight. The bacteria were diluted into 4.5 mL of media with antibiotics to reach an optical density of 0.05 measured at 550 nm.

Off-gas analysis of isoprene was performed using a gas chromatograph-mass spectrometer (GC-MS) (Agilent) headspace assay. Sample preparation was as follows: 100 μL of whole broth was placed in a sealed GC vial and incubated at 30° C. for a fixed time of 30 minutes. Following a heat kill step, consisting of incubation at 70° C. for 5 minutes, the sample was loaded on the GC.

Optical density (OD) at a wavelength of 550 nm was obtained using a microplate reader (Spectramax) during the course of the run. Specific productivity was obtained by dividing the isoprene concentration (μg/L) by the OD reading and the time (hour).

The two strains EWL256 and RM11608-2 were assessed at 200 and 400 μM IPTG induction levels. Samples were analyzed for isoprene production and cell growth (OD₅₅₀) at 1, 2.5, 4.75, and 8 hours post-induction. Samples were done in duplicate.

(b) Results

The experiment demonstrated that at 2 different concentrations of IPTG the strain expressing the ybhE (pgl) had a dramatic 2-3 fold increase in specific productivity of isoprene compared to the control strain.

ii) Isoprene Fermentation from E. coli Expressing Cm-GI1.2-KKDyI, M. mazei Mevalonate Kinase, P. alba Isoprene Synthase, and ybhE (pgl) (RM111608-2) and Grown in Fed-Batch Culture at the 15-L Scale

Medium Recipe (per liter fermentation medium): K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified trace Metal Solution: Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micron filter

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the upper mevalonic acid (MVA) pathway (pCL Upper), the integrated lower MVA pathway (gi1.2KKDyI), high expression of mevalonate kinase from M. mazei and isoprene synthase from P. alba (pTrcAlba-mMVK), and high expression of E. coli pgl (pBBR-pgl). This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 34° C. A frozen vial of the E. coli strain was thawed and inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 15-L bioreactor bringing the initial volume to 5-L.

Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 40 hour (59 hour) fermentation was 3.1 kg (4.2 kg at 59 hour). Induction was achieved by adding IPTG. The IPTG concentration was brought to 110 μM when the optical density at 550 nm (OD₅₅₀) reached a value of 4. The IPTG concentration was raised to 192 μM when OD₅₅₀ reached 150. The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 164A. The isoprene level in the off gas from the bioreactor was determined using a Hiden mass spectrometer. The isoprene titer increased over the course of the fermentation to a maximum value of 33.2 g/L at 40 hours (48.6 g/L at 59 hours) (FIG. 164B). The isoprene titer increased over the course of the fermentation to a maximum value of 40.0 g/L at 40 hours (60.5 g/L at 59 hours) (FIG. 164C). The total amount of isoprene produced during the 40-hour (59-hour) fermentation was 281.3 g (451.0 g at 59 hours) and the time course of production is shown in FIG. 164D. The time course of volumetric productivity is shown in FIG. 164E and shows that an average rate of 1.0 g/L/hr was maintained between 0 and 40 hours (1.4 g/L/hour between 19 and 59 hour). The metabolic activity profile, as measured by CER, is shown in FIG. 164F. The molar yield of utilized carbon that went into producing isoprene during fermentation was 19.6% at 40 hours (23.6% at 59 hours). The weight percent yield of isoprene from glucose was 8.9% at 40 hours (10.7% at 59 hours).

Example 25 Co-Production of Isoprene and Hydrogen in E. coli Strains Expressing M. mazei Mevalonate Kinase, P. alba Isoprene Synthase, pCL Upper MVA (E. faecalis mvaE and mvaS) and ybhE (pgl)

Collection and Analysis of Fermentation Off-Gas for Hydrogen and Isoprene Levels

Fermentations were performed using strains RM111608-2 (E. coli BL21 (DE3), pCL

Upper MVA, cmR-gi1.2-yKKDyI, pTrcAlba-mMVK, pBBR cmR-gi1.5-pgl) and EWL 256 (E. coli BL21 (DE3), pCL Upper MVA, cmR-gi1.2-yKKDyI, pTrcAlba-mMVK). Construction of bacterial strains is described in Example 23 above.

Large scale production of isoprene from E. coli was determined from a fed-batch culture of E. coli strains EWL256 and RM111608-2 expressing M. mazei mevalonate kinase, P. alba isoprene synthase, pCL Upper MVA (E. faecalis mvaE and mvaS) and either constitutively expressing ybhE (pgl) (RM111608-2) or normally expressing ybhE (pgl) (EWL256). This experiment demonstrates that growing cells in the presence of glucose resulted in the co-production of isoprene and hydrogen.

The recipe for the fermentation medium (TM2) per liter of TM2 fermentation medium was as follows: K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 5 g, 1000× Modified Trace Metal Solution 1 ml. 1000× Modified Trace Metal Solution: Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. For the 1000× Modified Trace Metal Solution, each component is dissolved one at a time in Di H₂O, pH to 3.0 with HCl/NaOH, then brought to final volume in distilled water and filter sterilized with a 0.22 micron (μm) filter (this solution is not autoclaved). For the TM2 fermentation medium, all of the components were added together, dissolved in diH₂O, the pH was adjusted to 6.8 with potassium hydroxide (KOH), q.s. to volume, and the medium was filter sterilized with a 0.22 micron (μm) filter. Glucose was sourced from Cargill as 99DE (dextrose equivalent), 71% DS (dry solids) syrup.

Fermentations were performed in 15-L bioreactors with E. coli strains EWL256 or RM111608-2, containing the upper mevalonic acid (MVA) pathway (pCL Upper MVA), the integrated lower MVA pathway (cmR-gi1.2-yKKDyI), mevalonate kinase from M. mazei and isoprene synthase from P. alba (pTrcAlba-mMVK), and constitutively expressing ybhE (pgl) (RM111608-2) or normally expressing ybhE (pgl) (EWL256). This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation conditions (pH 7.0 and temperature 34° C.).

An inoculum of the appropriate E. coli strain taken from a frozen vial was prepared in peptone-yeast extract medium. After the inoculum grew to OD₅₅₀=0.6, 600 mL was used to inoculate a 15-L bioreactor containing TM2 medium. Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 67 hour fermentation was 3.9 kg. Induction was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The IPTG concentration was brought to 102 μM when the optical density at 550 nm (OD₅₅₀) reached a value of 9. The IPTG concentration was raised to 192 μM when OD₅₅₀ reached 140. At various times after inoculation, samples were removed and the amount of isoprene produced was determined as described below. Levels of hydrogen, nitrogen, oxygen, carbon dioxide, and isoprene in the off gas from the bioreactor were determined using a Hiden HPR-20 mass spectrometer as discussed below.

Samples of fermentation off-gas from 15-L bioreactors were collected into 20 mL glass headspace vials by sparging the vials at 1 L_(offgas)/min for 10 seconds and sealed with metal screw caps fitted with teflon-coated septa (Agilent, CA). The vials were analyzed within 30 minutes of collection.

Analysis of the two samples was performed by infusion into a Hiden HPR-20 mass spectrometer (Hiden Analytics, U.K.) at a rate of 4 scc/min (4 mL/min) by placing the inlet tube of the mass spectrometer into the uncapped headspace vials for 1-2 minutes. The HPR-20 instrument was configured to scan masses corresponding to hydrogen (m/z 2), nitrogen (m/z 28), oxygen (m/z 32), carbon dioxide (m/z 44) and isoprene (m/z 67). The Faraday detector was used for masses 28, 32, 44 and 67. The SEM detector was used for hydrogen (m/z 2). Detector response was measured in arbitrary units of pressure (Torr). Absolute hydrogen levels were estimated by comparison to an authentic hydrogen gas standard. Results were recorded using MASsoft V 6.21.0.51 software (Hiden Analytics, United Kingdom).

Results

Off-gas samples were taken from two fermentation runs and analyzed as described above:

A) Strain RM111608-2 (E. coli BL21 (DE3), pCL upper, cmR-gi1.2-yKKDyI, pTrcAlba-mMVK, pBBR cmR-gi1.5-pgl). Sample was taken at 64.8 hours into the run during which time the fermentation was being run anaerobically with a nitrogen sparge at 1 vvm.

B) Strain EWL256 (E. coli BL21 (DE3), pCL upper, cmR-gi1.2-yKKDyI, pTrcAlba-mMVK). Sample was taken at 34.5 hours into the run during which time the fermentation was being run aerobically with an air sparge at 1 vvm.

The results are depicted in FIGS. 165A-B. In both cases low levels of hydrogen were detected, in addition to isoprene, oxygen and carbon dioxide. The baseline reading for hydrogen was 0.95×10⁻⁸ Ton. Both Sample A and B gave reading of around 1.3×10⁻⁸ Ton. Based on a comparison to a hydrogen standard, the amount of hydrogen present in the off-gas for samples A and B was estimated to be less than 10 ppmv (parts per million volume) but above the baseline. As shown in FIGS. 165A-B, both samples A and B also contained significant amounts of isoprene and carbon dioxide.

Example 26 Co-Production of Isoprene and Hydrogen in E. coli Strains Expressing M. mazei Mevalonate Kinase, P. alba Isoprene Synthase, pCL Upper MVA (E. faecalis mvaE and mvaS) and ybhE (pgl)

Collection and Analysis of Fermentation Off-Gas for Hydrogen and Isoprene Levels

The objective of this experiment is co-produce hydrogen and isoprene in an engineered strain of E. coli. For this purpose, a portion of the hyc operon encoding E. coli hydrogenase-3 will be expressed in strain EWL256 [BL21 (DE3), pCL upper, cmR-gi1.2-yKKDyI, pTrcAlba-mMVK], prepared as described herein, although any of the bacterial strains described herein, such as RM111608-2, can be similarly modified. An expression construct comprising hyc operon genes hycB (gi|16130631), hycC (gi|16130630), hycD (gi|16130629), hycE (gi|16130628), hycF (gi|16130627), and hycG (gi|16130626) is prepared by standard cloning methods known in the art based upon publicly available gene sequences, and introduced into strain EWL256 to produce new strain EWL256+Hyd-3.

The impact of additional mutations on co-production of hydrogen and isoprene is assessed alone or in combination in EWL256+Hyd-3, by introducing genes involved in the maturation or regulation of hydrogenase-3 (e.g., hycH (gi|16130625) and hycI (gi|16130624)), by inactivating or deleting genes involved in hydrogen uptake or transport (e.g., E. coli hydrogenase-1 (hya operon) and hydrogenase-2 (hyb operon)) or related proteins (e.g., formate dehydrogenase (fdhF (gi|16130624)), repressor of formate lyase (hycA (gi|16130632)), formate dehydrogenase N, alpha subunit (fdnG (gi|16129433)), formate dehydrogenase 0, large subunit (fdoG (gi|16131734)), nitrate reductase (narG (gi|16129187)), fumarate reductase regulator (fnr (gi|16129295)), and acetyl-coenzyme A synthetase (acs (gi|16131895))), by activating genes involved in upregulation of hydrogenases (e.g., activator of formate hydrogen lyase (fhlA (gi|16130638)), by inactivating or deleting genes involved in the production of fermentation side products (e.g., lactate dehydrogenase (ldhA (gi|16129341)), fumarate reductase membrane protein (frdC (gi|16131977)), alcohol dehydrogenase (adhE (gi|16129202)), pyruvate oxidase (poxB (gi|16128839)), pyruvate dehydrogenase E1 component ackA/pta (aceE (gi|16128107)), formate dehydrogenase regulatory protein (hycA (gi|16130632)), and formate transporters A and B (FocA (gi|16128871) and FocB (gi|16130417)), or by expression of heterologous genes involved in hydrogen metabolism (e.g., glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum (gapC (gi|15893997)).

Fermentations are performed using engineered variants of strain EWL 256+Hyd-3 (BL21 (DE3), pCL upper, cmR-gi1.2-yKKDyI, pTrcAlba-mMVK and hycB-F), modified to comprise one or more additional mutations as described herein, either alone or in combination, essentially as described in Example 25 above. Co-production of hydrogen and isoprene is assessed by analysis of off-gas samples essentially as described above. Strains are selected for further analysis based upon the rate of isoprene and hydrogen co-production.

Unless defined otherwise, the meanings of all technical and scientific terms used herein are those commonly understood by one of skill in the art to which this invention belongs. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) provide one of skill with a general dictionary of many of the terms used in this invention. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary. One of skill in the art will also appreciate that any methods and materials similar or equivalent to those described herein can also be used to practice or test the invention.

Example 27 Production of Isoprene or Mevalonate from Fatty Acid or Palm Oil in E. coli fadR atoC LS5218 Containing the Upper or Upper and Lower Mevalonic Acid Pathway Plus Kudzu Isoprene Synthase

Escherichia coli fadR atoC strain LS5218 (#6966) was obtained from the Coli Genetic Stock Center. FadR encodes a transcription repressor that negatively regulates expression of the genes encoding fatty acid degradation enzymes (Campbell et al., J. Bacteriol. 183: 5982-5990, 2001). AtoC is a response regulator in a two-component regulatory system wherein AtoS regulates acetolactate metabolism. The fadR atoC strain allows constitutive expression of the fatty acid degradation genes and incorporates long chain fatty acids into long-chain-length polyhydroxyalkanoates. When palm oil is used as a carbon source for either mevalonate or isoprene production, the palm oil was converted to glycerol plus fatty acid. Methods for this are well known in the art, and it can be done either enzymatically by incubation with a lipase (for example Porcine pancreatic lipase, Candida rugosa lipase, or other similar lipases) or chemically by saponification with a base such as sodium hydroxide.

i) E. coli fadR atoC Strain Expressing the Upper Mevalonic Acid Pathway

Strain WW4 was created by electroporating pCLPtrcUpperPathway into LS5218 using standard methods (Sambrooke et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989). Incorporation of the plasmid was demonstrated by the production of mevalonic acid (MVA) when cells were cultured in TM3 medium supplemented with either C12 fatty acid (FA) or palm oil as the carbon source. To demonstrate production of MVA by WW4 from fatty acid, cells from an overnight culture were diluted 1 to 100 into 5 mL of modified TM3 medium (TM3 without yeast extract) supplemented with 0.25% C12 FA (Sigma cat #L9755). The first sign of MVA production (24 mg/L) was apparent after overnight incubation at 30° C. of the IPTG induced culture. Production increased over three days with the final level of 194 mg/L of MVA produced. To demonstrate production of MVA by WW4 from oil, cells from an overnight culture were diluted 1 to 100 into modified TM3 medium supplemented with 200 mg of digested palm oil per 5 mL of TM3 medium. The first sign of MVA production (50 mg/L) was apparent after overnight incubation of the IPTG induced culture at 30° C. Production increased over three days with a final level of 500 mg/L of MVA produced.

ii) E. coli fadR atoC Strain Expressing the Upper and Lower MVA Pathway Plus Kudzu Isoprene Synthase

Escherichia coli strain WW4 (LS5218 fadR atoC pCLPtrcUpperPathway) was transformed with pMCM118 [pTrcKKDyIkIS] to yield WW10. The incorporation of the plasmid was demonstrated by evidence of production of isoprene when the strain was cultured in TM3 and glucose and induced with IPTG (100, 300, or 900 μM). The strain was relatively sensitive to IPTG and showed a significant growth defect even at 100 μM IPTG. These results are shown in FIG. 70A.

To test isoprene production from dodecanoic acid, WW10 was cultured overnight in L broth containing spectinomycin (50 μg/ml), and kanamycin (50 μg/ml) at 37 C with shaking at 200 rpm. The cells were washed with modified TM3 medium by centrifugation and resuspension in their original culture volume with this medium. The washed and resuspended cells from this starter culture were diluted 1 to 100 and 1 to 10 into 5 mL of modified TM3 medium containing 0.125% C12 Fatty Acid (Sigma cat #L9755).

To demonstrate production of mevalonate from palm oil, the oil was predigested with lipase at 37° C. and 250 rpm for several days to release the fatty acids (evidence of hydrolysis was judged by the foam formed when tubes were shaken).

In addition, a culture was set up by diluting the washed cells at 1 to 10 into modified TM3 medium contained in test tubes with palm oil. A further tube was set up by the addition of 0.125% C12FA to the remainder (2.5 mL) of the washed cells without further dilution (bioconversion). After 3.75 hours of growth at 30° C. with shaking at 250 rpm all of the cultures were induced by the addition of 50 μM IPTG. Incubation was continued for 4 hours after which time 200 μL of each of the cultures was assayed for isoprene accumulation with a modified head space assay (1 hour accumulation at 30° C. with shaking at 500 rpm). An additional isoprene assay was conducted by a 12 hour incubation of the assay glass block prior to GCMS analysis. Incubation of the induced cultures was continued overnight and 200 μL aliquots were again assayed for isoprene production (1 hour, 30 deg, 500 rpm Shel-Lab shaker) the following morning. Analysis of these cultures showed the production of significant levels of isoprene. The highest levels of isoprene were observed in the culture which was seeded at 1/10 dilution from the overnight starter culture after it had been incubated and induced overnight. This result suggests that this culture continued to grow and increase in cell density. These results are shown in FIG. 70B. Cell density could not be measured directly because the fatty acid suspension had a turbid appearance. Cell density of this culture was therefore determined by plating an aliquot of the culture and showed 8×10⁷ colony forming units. This corresponds approximately to an OD₆₀₀ of 0.1. Nevertheless, this culture provided significant isoprene production; no isoprene is observed for similar strains without the pathway described in this example.

Example 28 Expression of Isoprene-Synthase from Plant in Streptomyces sp.

The gene for isoprene synthase Kudzu was obtained from plasmid pJ201:19813. Plasmid pJ201:19813 encodes isoprene synthase from Pueraia lobata (Kudzu plant) and was codon-optimized for Pseudomonas fluorescens, Pseudomonas putida, Rhodopseudomonas palustris and Corynebacterium (FIGS. 79A-79C (SEQ ID NO:123)). Digestion of plasmid pJ201:19813 with restriction enzymes NdeI and BamHI liberated gene iso19813 that was ligated into the Streptomyces-E. coli shuttle vector pUWL201PW (Doumith et al., Mol. Gen. Genet. 264: 477-485, 2000; FIG. 71) to generate pUWL201_iso. Successful cloning was verified by restriction analysis of pUWL201_iso. Expression of isoprene synthase iso19813 was under control of the erm-promoter which allows for constitutive expression in Streptomycetes species, but not for expression in E. coli.

PUWL201PW (no insert) and pUWL201 iso were introduced in Streptomyces albus J1074 (Sanchez et al., Chem. Biol. 9:519-531, 2002) by transformation of protoplasts as described by Hopwood et al., The John innes foundation, Norwich, 1985.

A 200 μl aliquot of protoplast suspensions was transformed with 1.9 ng pUWL201PW or 2.9 ng pUWL201_iso. After incubation overnight at 28° C. on non-selective R5-agarplates, positive transformants were selected by further incubation for 4 days in R3-overlay agar containing thiostrepton (250 μg/ml). Thiostrepton resistant transformants were examined for presence of the pUWL-plasmids by plasmid preparation using Plasmid Mini Kit (Qiagen). Prepared plasmid DNA was reintroduced in E. coli DH5α to generate sufficient amounts of plasmid DNA to be analyzed by restriction analysis. Positive transformants were selected on ampicillin-containing L-agar plates and insert analysis was done by digestion of plasmid DNA with NdeI and BamHI endonucleases. Isoprene synthase was identified as a 1.7 kb fragment in positive pUWL201 iso clones while in the control strains (pUWL201PW) no such fragment was observed.

Wild type strain and transformants of S. albus containing control plasmid pUWL201PW or isoprene synthase encoding pUWL201 iso were analyzed for isoprene formation. Strains were cultivated in duplicate on solid media (tryptic soy broth agar, TSB; 2.5 ml) in presence or absence of thiostrepton (200 ng/ml) and incubated for 4 days at 28° C. in sealed head-space vials (total volume 20 ml). 500 μl head-space samples (end point measurements) were analyzed by GC-MS in SIM-mode and isoprene was identified according to reference retention times and molecular masses (67 m/z). Isoprene present in head-space samples was quantified by previously generated calibration curves. While wild-type S. albus and control strains harboring pUWL201PW produced isoprene in concentrations slightly higher than the detection limit (0.04-0.07 ppm), S. albus harboring pUWL201 iso produced isoprene in at least tenfold excess compared to controls (0.75 ppm; FIG. 72). The results demonstrate successful expression of plant-derived isoprene synthase in a prokaryotic organism of the Actinomycetes group.

Example 29 Recovery of Bioisoprene™

Bioisoprene™ was recovered from a set of four 14-L scale fermentations in a two-step operation involving stripping of isoprene from the fermentation off-gas stream by adsorption to activated carbon, followed by off-line steam desorption and condensation to give liquid Bioisoprene™ (FIGS. 166A and 166B). The total amount of Bioisoprene™ produced by the four fermentors was 1150 g (16.9 mol), of which 953 g (14 mol, 83%) was adsorbed by the carbon filters. Following the steam desorption/condensation step, the amount of liquid Bioisoprene™ recovered was 810 g, corresponding to an overall recovery yield of 70%. The recovered Bioisoprene™ was analyzed for the presence of impurities.

Analysis and Impurity Profile of Bioisoprene™ Liquid

Recovered Bioisoprene™ liquid was analyzed by GC/MS and gas chromatography/flame ionization detection (GC/FID) to determine the nature and levels of impurities. The product was determined to be >99.5% pure and contained several dominant impurities in addition to many minor components. The GC/FID chromatogram is depicted in FIG. 167, and the typical levels of impurities are shown in Table 19. The impurity profile was similar to other Bioisoprene™ batches produced on this scale.

TABLE 19 Summary of the nature and levels of impurities seen in several batches of Bioisoprene ™. Retention Time (min) Compound GC/MS GC/FID Conc. Range Ethanol 1.59 11.89 <50 ppm Acetone 1.624 12.673 <100 ppm Methacrolein 1.851 15.369 <200 ppm Methyl vinyl ketone 1.923 16.333 <20 ppm Ethyl acetate 2.037 17.145 100 to 800 ppm 3-Methyl-1,3- 2.27 18.875 50 to 500 ppm pentadiene Methyl vinyl oxirane 2.548 19.931 <100 ppm Isoprenol 2.962 21.583 <500 ppm 3-methyl-1-butanol 2.99 21.783 <50 ppm 3-hexen-1-ol 4.019 24.819 <100 ppm Isopentenyl acetate 4.466 25.733 200 to 1000 ppm 3-hexen-1-yl acetate 5.339 27.223 <400 ppm limonene 5.715 27.971 <500 ppm Other cyclics 5.50-6.50 27.5-28.0 <200 ppm Purification of Bioisoprene™ by Treatment with Adsorbents

Adsorbents are widely used by industry for the removal of trace impurities from hydrocarbon feedstocks. Suitable adsorbents include zeolite, alumina and silica-based materials. Bioisoprene™ can be substantially purified by passage over silica gel, and to a lesser extent with alumina. FIG. 168 shows the GC/FID chromatograms of a Bioisoprene™ sample before (A) and after treatment with alumina (B) or silica (C). The Selexsorb™ adsorbent products from BASF is one of the adsorbents of choice for the removal of polar impurities from Bioisoprene™ Specifically, the Selexsorb CD and CDX products are preferred given their proven utility for removal of polar impurities from isoprene and butadiene feedstocks.

Example 30 Chemical Transformations of Bioisoprene™

Chemicals and solvents were used as received from Sigma Aldrich Corp (WI, USA). BioIsoprene™ was produced by fermentation of E. coli BL21 strains expressing Isoprene synthase and a heterologous mevalonic acid (MVA) isoprene precursor biosynthetic pathway. Bioisoprene was recovered from fermentation off-gas by adsorption to activated carbon, followed by steam desorption and condensation to obtain crude, liquid Bioisoprene. Bioisoprene was purified by fractional distillation immediately before use.

¹H NMR Analysis

Proton (¹H) nuclear magnetic resonance (NMR) spectra were recorded on a Varian VNMRS 500 MHz NMR system. All NMR spectra are referenced to tetramethylsilane (TMS, 0 ppm) or chloroform (CHCl₃, 7.26 ppm) and peak frequencies are recorded in ppm unless otherwise specified. Samples were run in either deuterated chloroform (CDCl₃) or methanol (CD₃OD).

GC/MS Analysis

The analysis was performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μm film thickness) was used for separation of analytes. The autosampler was set up to inject 1 μL of a liquid sample from a 10 μL liquids syringe. The GC/MS method utilized helium as the carrier gas at a flow of 1 mL/minute. The injection port was held at 250° C. with a split ratio of 100:1. The oven program began at 50° C. for 2 minutes, increasing to 225° C. at a rate of 25° C./min. followed by a 1 minute hold for a total run time of 10 minutes. The Agilent 5793N mass selective detector was run in scan mode from m/z 29 to 500. A solvent delay of 1.5 minutes was employed. Under these conditions isoprene (2-methyl-1,3-butadiene) was observed to elute at 0.675 minutes.

GC/FID Analysis

The analysis was performed using an Agilent 6890 GC/FID system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in liquids mode. An Agilent DB-Petro GC column (100 m×0.25 mm; 0.50 μm film thickness) was used for separation of analytes. The autosampler was set up to inject 1 μL of a liquid sample from a 10 μL liquids syringe. The GC/FID method utilized helium as the carrier gas at a flow of 1 mL/minute. The injection port was held at 200° C. with a split ratio of 50:1. The oven program began at 50° C. for 15 minutes, increasing to 250° C. at a rate of 25° C./min. followed by a 10 minute hold for a total run time of 33 minutes. The FID detector was held at 280° C. in Constant makeup mode with a hydrogen flow of 35 mL/min and air flow of 250 mL/min. Under these conditions isoprene (2-methyl-1,3-butadiene) was observed to elute at 13.54 minutes.

I. Preparation of Cyclic Dimers of Isoprene through Thermal Diels-Alder Cycloaddition

Isoprene (1.02 g, 0.015 mole) was heated at 150° C. for 24 hours in the presence of 2,6-di-tert-butyl-4-methylphenol (0.165 g, 0.05 mole) acting as an inhibitor of thermal polymerization. The reaction was carried out in a sealed thick-walled glass reaction vessel that was dried in the vacuum oven for 24 hours prior to the reaction. The reaction mixture was stirred with a magnetic stirrer. The progress of the reaction was monitored by gas chromatography with mass-spectrometer detector (GC-MS). After the reaction was finished, the resulting mixture of isomers was purified using silica gel chromatography with hexane as the eluent. Following concentration on a rotary vacuum evaporator the product was characterized by GC-MS and ¹H-NMR. GC-MS: product A: 5.17, 5.19 min; product B: 5.72, 5.74 min; product C, 6.11 min. ¹H-NMR (CDCl₃) d: 5.8 (m); 5.4 (m); 4.95 (m); 2.4-1.2 (m).

II. Preparation of Isoprene Oligomers through Pd-Catalyzed Oligomerization

Isoprene (2.03 g, 0.03 mole) was mixed with isopropanol (1.79 g, 0.03 mole) in a sealed thick-walled glass reaction vessel. Prior to the reaction the glass chamber was dried in a vacuum oven for 24 hours. Transfer of all reagents was done under inert nitrogen atmosphere. Palladium acetylacetonate (0.55 mg, 0.06 mmole) and triphenylphosphine (1.49 mg, 0.19 mmol) were added to the reaction mixture. The reaction chamber was then heated to 100° C. for 24 hours with mixing provided via magnetic stirrer. The course of the reaction was analyzed by GC-MS. Once the reaction was finished the products were isolated by silica gel column chromatography using hexane as the eluent. The final products were characterized by GC-MS and ¹H-NMR spectroscopy. GC-MS: product A: 5.54 min; product B: 6.6 min; product C, 6.85 min.

III. Hydrogenation of Unsaturated Compounds

The mixture of isomers obtained in Example 2 (1.36 g, 0.01 mole) is put in a glass chamber equipped with magnetic stirrer and containing a hydrogenation catalyst (Pd/C, 5 wt %) (0.21 g, 0.1 mmol). All glassware is vacuum dried prior to carrying out the experiments. Hydrogen gas is introduced into the system and the pressure is kept at 3 atm. After several hours the reaction mixture Pd/C is filtered from the reaction mixture and the products are separated using silica gel chromatography. Final analysis is done using GC-MS and NMR.

IV. Preparation of Ethoxylated Derivatives of Isoprene

Isoprene (0.982 g, 0.014 mole) was mixed with absolute ethanol (0.665 g, 0.014 mol) in a thick glass wall chamber equipped with magnetic stirrer. A catalytic amount of concentrated sulfuric acid was added to the reaction mixture, followed by stirring overnight at 85° C. The progress of the reaction was monitored by GC-MS. After 16 hours of heating the GC-MS trace revealed the presence of a mixture of isomers with following retention times: product A: 2.66 min m/z⁺=99; product B: 3.88 m/z⁺=99, 114; product C, 5.41 min, m/z⁺=87, 99, 114.

V. Conversion of Isoprene to C10 Cyclic Dimers Using a Ruthenium Catalyst

The conversion of isoprene into C10 cyclic dimers has been achieved in excellent yield using a ruthenium catalyst to a mixture of dimethyl-cyclooctadienes (Itoh, Kenji; Masuda, Katsuyuki; Fukahori, Takahiko; Nakano, Katsumasa; Aoki, Katsuyuki; Nagashima, Hideo, Organometallics (1994), 13(3), 1020-9.) Conversions higher than 95% are reported at very moderate temperatures (as low as 60° C.). The catalyst, a pentamethylcyclopentadienyl-based ruthenium organometallic compound, can be prepared in two easy steps using ruthenium chloride (RuCl₃) and pentamethylcyclopentadiene (C₅Me₅H) as starting materials. For the dimerization reaction the catalyst is activated with silver triflate (AgOTf) to produce the active species, but other activators could be used as well.

VI. Conversion of Isoprene to C15 Trimers Using a Chromium Catalyst

Conversion of isoprene into the C15 trimers can be carried out using a chromium catalyst with a P—N—P ligand, N,N-bis(diarylphosphino)amine. The catalyst is prepared in situ and it is activated using MAO, as described in Bowen, L.; Charernsuk, M.; Wass, D. F. Chem. Commun. (2007) 2835-2837. The reported conversion of isoprene into the C15 trimers, consisting in a mixture (3:1) of linear and cyclic products, is as high as 95% at moderate temperatures (70° C.). Tetramers are identified as the major other product in every case.

VII. Hydrogenation of Isoprene

Isoprene (10 mL of a 10% solution in absolute ethanol (v/v)) was hydrogenated to 2-methylbutane (isopentane) in a continuous manner using an H-cube hydrogenation instrument (ThalesNano, Princeton, N.J., U.S.A.). The isoprene solution was pumped at 0.5 mL/min through a 10% Pd/C catalyst cartridge held at 70° C. Hydrogen gas was introduced using “full mode” at 1 atm pressure. The product was collected and analyzed by ¹H NMR and GC/FID which confirmed the conversion of isoprene to 2-methylbutane in over 90% yield, in addition to minor amounts of partially hydrogenated mono-olefins. ¹H NMR (500 MHz, CDCl₃): δ0.8 (m, 9H, CH₃); 1.12 (m, 2H, CH₂); 1.37 (m, 1H, CH). GC/FID: 2-methylbutane; retention time=12.69 minutes.

VIII. Partial Hydrogenation of Isoprene

BioIsoprene™ product (50 mL, 0.5 mol) was mixed with toluene (200 mL) and partially hydrogenated over a 5% Pd/C catalyst on an Midi-Cube hydrogenation instrument (ThalesNano, Budapest, Hungary) at 40° C. and 5 bar hydrogen pressure. Substrate flow rate was 10 mL/min and hydrogen was delivered at 125 mL/min (5 mmol/min). The product stream was recycled through the instrument for a period of 2 hours after which time an aliquot of the product was analyzed by GC/MS and GC/FID which showed that the majority of the starting material had been converted to a mixture of isoamylenes (2-methyl-1-butene, 2-methyl-2-butene and 3-methyl-1-butene), in addition to isopentane and some unreacted isoprene (FIG. 170).

IX. Selective Hydrogenation of BioIsoprene™ Product

BioIsoprene™ product is selectively hydrogenated under the conditions cited in the above example using an eggshell Pd/d-Al₂O₃ catalyst giving a mixture of isoamylenes where 2-methyl-2-butene is the dominant product accounting for >50% of the total isoamylenes and 3-methyl-1-butene is the minor product accounting for <25% of the total isoamylene products as determined by GC/MS analysis. The amount of isopentane and residual isoprene account for <10% of the total product stream. A similar result is obtained when a sulfided palladium on carbon catalyst is used to perform the reaction.

X. Partial Hydrogenation of BioIsoprene™ Product in the Gas Phase

A dry gas stream containing BioIsoprene™ product is mixed with a slight excess of hydrogen gas (mol/mol) and the gaseous mixture passed over a heterogenous hydrogenation catalyst, such as a Group IB-promoted palladium catalyst with high pore volume as described in US Pat. Appl. 20090203520, to produce a mixture of isoamylenes and one or more impurities derived from the fermentation process from which the BioIsoprene™ product was originally derived. The conversion is carried out at pressures ranging from 0.5 to 200 bar, and temperatures from 0° C. to 200° C.

XI. Dimerization of Isoamylenes with a Solid Acid Catalyst

2-Methyl-2-butene (1.5 mL) and toluene (4 mL) were stirred at room temperature with Amberlyst 15 acid resin (186 mg) for 12 h at room temperature. An aliquot (500 uL) was removed from the reaction mixture and transferred to a GC vial. Analysis of the mixture was performed by GC/MS (FIG. 171) and revealed the partial conversion to C10 dimers (diisoamlylenes) suitable as BioIsofuel™ components.

The products of the dimerization reaction (diisoamylenes, C10 dimate) are optionally fully hydrogenated under conditions described in Example 30, part VII, or through conditions known in the art for the hydrogenation of olefins to isoparaffins [for example, see Marichonna (2001)].

XII. Oligomerization of BioIsoprene™ Product with a Solid Acid Catalyst

A mixture of BioIsoprene™ monomer, 2-methyl-2-butene (1.5 mL) and toluene (4 mL) was stirred at room temperature with Amberlyst 15 acid resin (186 mg) for 12 h at room temperature. An aliquot (500 uL) was removed from the reaction mixture and transferred to a GC vial. Analysis of the mixture was performed by GC/MS (FIG. 172) and revealed a complex mixture of products consisting of isoprene, linear, cyclic and aromatic C10, C15 and higher oligomers.

XIII. Continuous Oligomerization of BioIsoprene™ Product with a Solid Acid Catalyst

BioIsoprene™ monomer is continuously converted into C10 dimers and C15 trimers in a dimerization reactor containing Amberlyst 15 ion exchange resin or an equivalent catalyst. The BioIsoprene™ feed stream comprises BioIsoprene™ monomer and optionally C5 derivatives of BioIsoprene™ and a co-solvent. The process is conducted at temperatures ranging from 20 to 200° C. and pressures from 0.5 to 200 bar. The products of the dimerization step are fractionated in a first fractionation column to separate unreacted isoprene from higher (>C5) oligomers. The C5 fraction is returned to the dimerization reactor and the heavy >C5 fraction is introduced into a second fractionation column in which the desired C10/C15 fraction is collected from the overhead stream. The bottom fraction consisting of >C15 oligomers is fed into a heavy recycle reactor containing a metathesis catalyst such as the Grubbs 2^(nd) generation catalyst. The metathesis catalyst converts a portion of the higher oligomer fraction into lighter components by olefin cross-metathesis reactions that are subsequently fed into fractionation column #1 as depicted in FIG. 173.

Overall, the process results in conversion of BioIsoprene monomer into C10 dimer and C15 trimer BioIsofuel™ precursors which are then subjected to partial or complete hydrogenation under conditions described in example 30, section VII. The resulting partially or fully saturated compounds are suitable as BioIsofuel™ compositions and as BioIsofuel™ blendstocks.

Example 31 ¹³C/¹²C Isotope Analysis

¹³C analysis can be done by loading 0.5 to 1.0 mg samples into tin cups for carbon isotopic analysis using a Costech ECS4010 Elemental Analyzer as an inlet for a ThermoFinnigan Delta Plus XP isotope ratio mass spectrometer. Samples are dropped into a cobaltous/cobaltic oxide combustion reactor at 1020° C. with combustion gases being passed in a helium stream at 85 mL/min through a copper reactor (650° C.) to convert NO_(x) to N₂. CO₂ and N₂ are separated using a 3-m 5 Å molecular sieve column. Then, ¹³C/¹²C ratios are calibrated to the VPDB scale using two laboratory standards (Acetanilide B, −29.52±0.02‰m and cornstarch A, −11.01±0.02‰) which have been carefully calibrated to the VPDB scale by off-line combustion and dual-inlet analysis using the 2-standard approach of T. B. Coplen et al, New Guidelines for d¹³C Measurements, Anal. Chem., 78, 2439-2441 (2006). The teachings of Coplen are incorporated herein by reference for the purpose of teaching the technique for determining d¹³C values.

U.S. Provisional Patent Application No. 61/133,521 filed on Jun. 30, 2008 and WO 2010/05525 A1 list δ¹³C values for feedstock and polymers of isoprene derived from various sources, including ones listed in Table 20.

TABLE 20 Sample δ¹³C Palm oil −30.00 Yeast extract −25.70 Commercial polyisoprene from extractive distillation −23.83 Sugar from softwood pulp −23.00 Polyisoprene from Isoprene Sample B (emulsion polymerization) −19.67 Invert Sugar −15.37 Polyisoprene from Isoprene Sample A (Neodymium catalyst) −14.85 Glucose from bagasse −13.00 Glucose from corn stover −11.20 Cornstarch −11.10 Glucose −10.73

Example 32 Exemplary Fuel Properties

Table 21 lists fuel properties of certain compounds that can be made from isoprene using methods described herein.

TABLE 21 Lower Higher Vapor Heating Heating Boiling pressure ΔHc Value Value MW point Density (Torr, (kcal/ (kBtu/ (kBtu/ Octane Formula (g/mol) (° C.) (g/mol³) 25° C.)* mol) gal) gal) (Cetane) a-Limonene C₁₀H₁₆

136.23 177 0.8477 1.54 1474 130.6 137.9  88 1-Methyl-4- isopropyl- cyclohexane C₁₀H₂₀

140.27 170.2 0.8060 2.16 1561 126.5 134.9  75 2,7-dimethyl-, (4E)-2,4,6- Octatriene C₁₀H₁₆

136.23 186.4* 0.782 0.915 1481 121.1 127.8 110 2,7-dimethyl- octane C₁₀H₂₂

142.28 160 0.728 3.35 1620 116.5 124.7  97 3,7-dimethyl- 1,5-cyclo- octadiene C₁₀H₁₆

136.23 182.7* 0.860 1.09 1490 134.0 141.4  95 1,5-Dimethyl- cyclooctene (BIF-10) C₁₀H₁₈

138.25 178.5* 0.830 1.33 1545 131.6 139.4  90 1,5-Dimethyl cyclooctane C₁₀H₂₀

140.27 158.5 0.800 1.39 1560 125.4 133.8  85 2,6,11- Trimethyl- dodecane C₁₅H₃₂

212.41 247.8* 0.766 0.0396 2400 121.7 130.1 (65) Cyclo- dodecane, 1,4,8- trimethyl C₁₅H₃₀

210.40 278.0* 0.850 7.38E−3 2380 135.7 144.6 (40) BIF-5 C₁₅H_(31.5) 3:1 mixture of linear and cyclic C15 211.9 255.4 0.787 0.0315 2395 125.2 133.7 (58.8)

The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole.

All publications, patent applications, and patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or patent were specifically and individually indicated to be incorporated by reference. In particular, all publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methodologies which might be used in connection with the invention. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

APPENDIX 1 Exemplary 1-deoxy-D-xylulose-5-phosphate synthase nucleic acids and polypeptides ATH: AT3G21500(DXPS1) AT4G15560(CLA1) HIQ: CGSHiGG_01080 AT5G11380(DXPS3) HDU: HD0441(dxs) OSA: 4338768 4340090 4342614 HSO: HS_0905(dxs) CME: CMF089C PMU: PM0532(dxs) PFA: MAL13P1.186 MSU: MS1059(dxs) TAN: TA20470 APL: APL_0207(dxs) TPV: TP01_0516 XFA: XF2249 ECO: b0420(dxs) XFT: PD1293(dxs) ECJ: JW0410(dxs) XCC: XCC2434(dxs) ECE: Z0523(dxs) XCB: XC_1678 ECS: ECs0474 XCV: XCV2764(dxs) ECC: c0531(dxs) XAC: XAC2565(dxs) ECI: UTI89_C0443(dxs) XOO: XOO2017(dxs) ECP: ECP_0479 XOM: XOO_1900(XOO1900) ECV: APECO1_1590(dxs) VCH: VC0889 ECW: EcE24377A_0451(dxs) VVU: VV1_0315 ECX: EcHS_A0491 VVY: VV0868 STY: STY0461(dxs) VPA: VP0686 STT: t2441(dxs) VFI: VF0711 SPT: SPA2301(dxs) PPR: PBPRA0805 SEC: SC0463(dxs) PAE: PA4044(dxs) STM: STM0422(dxs) PAU: PA14_11550(dxs) YPE: YPO3177(dxs) PAP: PSPA7_1057(dxs) YPK: y1008(dxs) PPU: PP_0527(dxs) YPM: YP_0754(dxs) PST: PSPTO_0698(dxs) YPA: YPA_2671 PSB: Psyr_0604 YPN: YPN_0911 PSP: PSPPH_0599(dxs) YPP: YPDSF_2812 PFL: PFL_5510(dxs) YPS: YPTB0939(dxs) PFO: Pfl_5007 YPI: YpsIP31758_3112(dxs) PEN: PSEEN0600(dxs) SFL: SF0357(dxs) PMY: Pmen_3844 SFX: S0365(dxs) PAR: Psyc_0221(dxs) SFV: SFV_0385(dxs) PCR: Pcryo_0245 SSN: SSON_0397(dxs) ACI: ACIAD3247(dxs) SBO: SBO_0314(dxs) SON: SO_1525(dxs) SDY: SDY_0310(dxs) SDN: Sden_2571 ECA: ECA1131(dxs) SFR: Sfri_2790 PLU: plu3887(dxs) SAZ: Sama_2436 BUC: BU464(dxs) SBL: Sbal_1357 BAS: BUsg448(dxs) SLO: Shew_2771 WBR: WGLp144(dxs) SHE: Shewmr4_2731 SGL: SG0656 SHM: Shewmr7_2804 KPN: KPN_00372(dxs) SHN: Shewana3_2901 BFL: Bfl238(dxs) SHW: Sputw3181_2831 BPN: BPEN_244(dxs) ILO: IL2138(dxs) HIN: HI1439(dxs) CPS: CPS_1088(dxs) HIT: NTHI1691(dxs) PHA: PSHAa2366(dxs) HIP: CGSHiEE_04795 PAT: Patl_1319 SDE: Sde_3381 HAR: HEAR0279(dxs) PIN: Ping_2240 MMS: mma_0331 MAQ: Maqu_2438 NEU: NE1161(dxs) MCA: MCA0817(dxs) NET: Neut_1501 FTU: FTT1018c(dxs) NMU: Nmul_A0236 FTF: FTF1018c(dxs) EBA: ebA4439(dxs) FTW: FTW_0925(dxs) AZO: azo1198(dxs) FTL: FTL_1072 DAR: Daro_3061 FTH: FTH_1047(dxs) TBD: Tbd_0879 FTA: FTA_1131(dxs) MFA: Mfla_2133 FTN: FTN_0896(dxs) HPY: HP0354(dxs) NOC: Noc_1743 HPJ: jhp0328(dxs) AEH: Mlg_1381 HPA: HPAG1_0349 HCH: HCH_05866(dxs) HHE: HH0608(dxs) CSA: Csal_0099 HAC: Hac_0968(dxs) ABO: ABO_2166(dxs) WSU: WS1996 AHA: AHA_3321(dxs) TDN: Tmden_0475 BCI: BCI_0275(dxs) CJE: Cj0321(dxs) RMA: Rmag_0386 CJR: CJE0366(dxs) VOK: COSY_0360(dxs) CJJ: CJJ81176_0343(dxs) NME: NMB1867 CJU: C8J_0298(dxs) NMA: NMA0589(dxs) CJD: JJD26997_1642(dxs) NMC: NMC0352(dxs) CFF: CFF8240_0264(dxs) NGO: NGO0036 CCV: CCV52592_1671(dxs) CCV52592_1722 CVI: CV_2692(dxs) CHA: CHAB381_1297(dxs) RSO: RSc2221(dxs) CCO: CCC13826_1594(dxs) REU: Reut_A0882 ABU: Abu_2139(dxs) REH: H16_A2732(dxs) NIS: NIS_0391(dxs) RME: Rmet_2615 SUN: SUN_2055(dxs) BMA: BMAA0330(dxs) GSU: GSU0686(dxs-1) GSU1764(dxs-2) BMV: BMASAVP1_1512(dxs) GME: Gmet_1934 Gmet_2822 BML: BMA10299_1706(dxs) PCA: Pcar_1667 BMN: BMA10247_A0364(dxs) PPD: Ppro_1191 Ppro_2403 BXE: Bxe_B2827 DVU: DVU1350(dxs) BUR: Bcep18194_B2211 DVL: Dvul_1718 BCN: Bcen_4486 DDE: Dde_2200 BCH: Bcen2424_3879 LIP: LI0408(dsx) BAM: Bamb_3250 DPS: DP2700 BPS: BPSS1762(dxs) ADE: Adeh_1097 BPM: BURPS1710b_A0842(dxs) MXA: MXAN_4643(dxs) BPL: BURPS1106A_A2392(dxs) SAT: SYN_02456 BPD: BURPS668_A2534(dxs) SFU: Sfum_1418 BTE: BTH_II0614(dxs) PUB: SAR11_0611(dxs) BPE: BP2798(dxs) MLO: mlr7474 BPA: BPP2464(dxs) MES: Meso_0735 BBR: BB1912(dxs) SME: SMc00972(dxs) RFR: Rfer_2875 ATU: Atu0745(dxs) POL: Bpro_1747 ATC: AGR_C_1351 PNA: Pnap_1501 RET: RHE_CH00913(dxs) AJS: Ajs_1038 RLE: RL0973(dxs) MPT: Mpe_A2631 BME: BMEI1498 BMF: BAB1_0462(dxs) GKA: GK2392 BMS: BR0436(dxs) GTN: GTNG_2322 BMB: BruAb1_0458(dxs) LMO: lmo1365(tktB) BOV: BOV_0443(dxs) LMF: LMOf2365_1382(dxs) BJA: bll2651(dxs) LIN: lin1402(tktB) BRA: BRADO2161(dxs) LWE: lwe1380(tktB) BBT: BBta_2479(dxs) LLA: L108911(dxsA) L123365(dxsB) RPA: RPA0952(dxs) LLC: LACR_1572 LACR_1843 RPB: RPB_4460 LLM: llmg_0749(dxsB) RPC: RPC_1149 SAK: SAK_0263 RPD: RPD_4305 LPL: lp_2610(dxs) RPE: RPE_1067 LJO: LJ0406 NWI: Nwi_0633 LAC: LBA0356 NHA: Nham_0778 LSL: LSL_0209(dxs) BHE: BH04350(dxs) LGA: LGAS_0350 BQU: BQ03540(dxs) STH: STH1842 BBK: BARBAKC583_0400(dxs) CAC: CAC2077 CA_P0106(dxs) CCR: CC_2068 CPE: CPE1819 SIL: SPO0247(dxs) CPF: CPF_2073(dxs) SIT: TM1040_2920 CPR: CPR_1787(dxs) RSP: RSP_0254(dxsA) RSP_1134(dxs) CTC: CTC01575 JAN: Jann_0088 Jann_0170 CNO: NT01CX_1983 RDE: RD1_0101(dxs) RD1_0548(dxs) CTH: Cthe_0828 MMR: Mmar10_0849 CDF: CD1207(dxs) HNE: HNE_1838(dxs) CBO: CBO1881(dxs) ZMO: ZMO1234(dxs) ZMO1598(dxs) CBA: CLB_1818(dxs) NAR: Saro_0161 CBH: CLC_1825(dxs) SAL: Sala_2354 CBF: CLI_1945(dxs) ELI: ELI_12520 CKL: CKL_1231(dxs) GOX: GOX0252 CHY: CHY_1985(dxs) GBE: GbCGDNIH1_0221 GbCGDNIH1_2404 DSY: DSY2348 RRU: Rru_A0054 Rru_A2619 DRM: Dred_1078 MAG: amb2904 PTH: PTH_1196(dxs) MGM: Mmc1_1048 SWO: Swol_0582 SUS: Acid_1783 CSC: Csac_1853 BSU: BG11715(dxs) TTE: TTE1298(dxs) BHA: BH2779 MTA: Moth_1511 BAN: BA4400(dxs) MPE: MYPE730 BAR: GBAA4400(dxs) MGA: MGA_1268(dxs) BAA: BA_4853 MTU: Rv2682c(dxs1) Rv3379c(dxs2) BAT: BAS4081 MTC: MT2756(dxs) BCE: BC4176(dxs) MBO: Mb2701c(dxs1) Mb3413c(dxs2) BCA: BCE_4249(dxs) MLE: ML1038(dxs) BCZ: BCZK3930(dxs) MPA: MAP2803c(dxs) BTK: BT9727_3919(dxs) MAV: MAV_3577(dxs) BTL: BALH_3785(dxs) MSM: MSMEG_2776(dxs) BLI: BL01523(dxs) MMC: Mmcs_2208 BLD: BLi02598(dxs) CGL: NCgl1827(cgl1902) BCL: ABC2462(dxs) CGB: cg2083(dxs) BAY: RBAM_022600 CEF: CE1796 BPU: BPUM_2159 CDI: DIP1397(dxs) CJK: jk1078(dxs) AVA: Ava_4532 NFA: nfa37410(dxs) PMA: Pro0928(dxs) RHA: RHA1_ro06843 PMM: PMM0907(Dxs) SCO: SCO6013(SC1C3.01) SCO6768(SC6A5.17) PMT: PMT0685(dxs) SMA: SAV1646(dxs1) SAV2244(dxs2) PMN: PMN2A_0300 TWH: TWT484 PMI: PMT9312_0893 TWS: TW280(Dxs) PMB: A9601_09541(dxs) LXX: Lxx10450(dxs) PMC: P9515_09901(dxs) CMI: CMM_1660(dxsA) PMF: P9303_15371(dxs) AAU: AAur_1790(dxs) PMG: P9301_09521(dxs) PAC: PPA1062 PMH: P9215_09851 TFU: Tfu_1917 PMJ: P9211_08521 FRA: Francci3_1326 PME: NATL1_09721(dxs) FAL: FRAAL2088(dxs) TER: Tery_3042 ACE: Acel_1393 BTH: BT_1403 BT_4099 SEN: SACE_1815(dxs) SACE_4351 BFR: BF0873 BF4306 BLO: BL1132(dxs) BFS: BF0796(dxs) BF4114 BAD: BAD_0513(dxs) PGI: PG2217(dxs) FNU: FN1208 FN1464 CHU: CHU_3643(dxs) RBA: RB2143(dxs) GFO: GFO_3470(dxs) CTR: CT331(dxs) FPS: FP0279(dxs) CTA: CTA_0359(dxs) CTE: CT0337(dxs) CMU: TC0608 CPH: Cpha266_0671 CPN: CPn1060(tktB_2) PVI: Cvib_0498 CPA: CP0790 PLT: Plut_0450 CPJ: CPj1060(tktB_2) DET: DET0745(dxs) CPT: CpB1102 DEH: cbdb_A720(dxs) CCA: CCA00304(dxs) DRA: DR_1475 CAB: CAB301(dxs) DGE: Dgeo_0994 CFE: CF0699(dxs) TTH: TTC1614 PCU: pc0619(dxs) TTJ: TTHA0006 TPA: TP0824 AAE: aq_881 TDE: TDE1910(dxs) TMA: TM1770 LIL: LA3285(dxs) PMO: Pmob_1001 LIC: LIC10863(dxs) LBJ: LBJ_0917(dxs) LBL: LBL_0932(dxs) SYN: sll1945(dxs) SYW: SYNW1292(Dxs) SYC: syc1087_c(dxs) SYF: Synpcc7942_0430 SYD: Syncc9605_1430 SYE: Syncc9902_1069 SYG: sync_1410(dxs) SYR: SynRCC307_1390(dxs) SYX: SynWH7803_1223(dxs) CYA: CYA_1701(dxs) CYB: CYB_1983(dxs) TEL: tll0623 GVI: gll0194 ANA: alr0599 Exemplary acetyl-CoA-acetyltransferase nucleic acids and polypeptides HSA: 38(ACAT1) 39(ACAT2) STM: STM3019(yqeF) PTR: 451528(ACAT1) SFL: SF2854(yqeF) MCC: 707653(ACAT1) 708750(ACAT2) SFX: S3052(yqeF) MMU: 110446(Acat1) 110460(Acat2) SFV: SFV_2922(yqeF) RNO: 25014(Acat1) SSN: SSON_2283(atoB) SSON_3004(yqeF) CFA: 484063(ACAT2) 489421(ACAT1) SBO: SBO_2736(yqeF) GGA: 418968(ACAT1) 421587(RCJMB04_34i5) ECA: ECA1282(atoB) XLA: 379569(MGC69098) 414622(MGC81403) ENT: Ent638_3299 414639(MGC81256) SPE: Spro_0592 444457(MGC83664) HIT: NTHI0932(atoB) XTR: 394562(acat2) XCC: XCC1297(atoB) DRE: 30643(acat2) XCB: XC_2943 SPU: 759502(LOC759502) XCV: XCV1401(thlA) DME: Dmel_CG10932 Dmel_CG9149 XAC: XAC1348(atoB) CEL: T02G5.4 T02G5.7 T02G5.8(kat-1) XOO: XOO1881(atoB) ATH: AT5G48230(ACAT2/EMB1276) XOM: XOO_1778(XOO1778) OSA: 4326136 4346520 VCH: VCA0690 CME: CMA042C CME087C VCO: VC0395_0630 SCE: YPL028W(ERG10) VVU: VV2_0494 VV2_0741 AGO: AGOS_ADR165C VVY: VVA1043 VVA1210 PIC: PICST_31707(ERG10) VPA: VPA0620 VPA1123 VPA1204 CAL: CaO19.1591(erg10) PPR: PBPRB1112 PBPRB1840 CGR: CAGL0L12364g PAE: PA2001(atoB) PA2553 PA3454 PA3589 SPO: SPBC215.09c PA3925 MGR: MGG_01755 MGG_13499 PAU: PA14_38630(atoB) ANI: AN1409.2 PPU: PP_2051(atoB) PP_2215(fadAx) PP_3754 AFM: AFUA_6G14200 AFUA_8G04000 PP_4636 AOR: AO090103000012 AO090103000406 PPF: Pput_2009 Pput_2403 Pput_3523 Pput_4498 CNE: CNC05280 PST: PSPTO_0957(phbA-1) PSPTO_3164(phbA-2) UMA: UM03571.1 PSB: Psyr_0824 Psyr_3031 DDI: DDB_0231621 PSP: PSPPH_0850(phbA1) PSPPH_2209(phbA2) PFA: PF14_0484 PFL: PFL_1478(atoB-2) PFL_2321 PFL_3066 TET: TTHERM_00091590 TTHERM_00277470 PFL_4330(atoB-2) PFL_5283 TTHERM_00926980 PFO: Pfl_1269 Pfl_1739 Pfl_2074 Pfl_2868 TCR: 511003.60 PEN: PSEEN3197 PSEEN3547(fadAx) ECO: b2224(atoB) PSEEN4635(phbA) ECJ: JW2218(atoB) JW5453(yqeF) PMY: Pmen_1138 Pmen_2036 Pmen_3597 ECE: Z4164(yqeF) Pmen_3662 Pmen_3820 ECS: ECs3701 PAR: Psyc_0252 Psyc_1169 ECC: c2767(atoB) c3441(yqeF) PCR: Pcryo_0278 Pcryo_1236 Pcryo_1260 ECI: UTI89_C2506(atoB) UTI89_C3247(yqeF) PRW: PsycPRwf_2011 ECP: ECP_2268 ECP_2857 ACI: ACIAD0694 ACIAD1612 ACIAD2516(atoB) ECV: APECO1_3662(yqeF) APECO1_4335(atoB) SON: SO_1677(atoB) APECO1_43352(atoB) SDN: Sden_1943 ECX: EcHS_A2365 SFR: Sfri_1338 Sfri_2063 STY: STY3164(yqeF) SAZ: Sama_1375 STT: t2929(yqeF) SBL: Sbal_1495 SPT: SPA2886(yqeF) SBM: Shew185_1489 SEC: SC2958(yqeF) SBN: Sbal195_1525 SLO: Shew_1667 Shew_2858 BXE: Bxe_A2273 Bxe_A2335 Bxe_A2342 SPC: Sputcn32_1397 Bxe_A4255 Bxe_B0377 Bxe_B0739 SSE: Ssed_1473 Ssed_3533 Bxe_C0332 Bxe_C0574 Bxe_C0915 SPL: Spea_2783 BVI: Bcep1808_0519 Bcep1808_1717 SHE: Shewmr4_2597 Bcep1808_2877 Bcep1808_3594 SHM: Shewmr7_2664 Bcep1808_4015 Bcep1808_5507 Bcep1808_5644 SHN: Shewana3_2771 BUR: Bcep18194_A3629 Bcep18194_A5080 SHW: Sputw3181_2704 Bcep18194_A5091 ILO: IL0872 Bcep18194_A6102 Bcep18194_B0263 CPS: CPS_1605 CPS_2626 Bcep18194_B1439 PHA: PSHAa0908 PSHAa1454(atoB) Bcep18194_C6652 Bcep18194_C6802 PSHAa1586(atoB) Bcep18194_C6874 PAT: Patl_2923 Bcep18194_C7118 Bcep18194_C7151 SDE: Sde_3149 Bcep18194_C7332 PIN: Ping_0659 Ping_2401 BCN: Bcen_1553 Bcen_1599 Bcen_2158 Bcen_2563 MAQ: Maqu_2117 Maqu_2489 Maqu_2696 Bcen_2998 Bcen_6289 Maqu_3162 BCH: Bcen2424_0542 Bcen2424_1790 CBU: CBU_0974 Bcen2424_2772 Bcen2424_5368 LPN: lpg1825(atoB) Bcen2424_6232 Bcen2424_6276 LPF: lpl1789 BAM: Bamb_0447 Bamb_1728 Bamb_2824 LPP: lpp1788 Bamb_4717 Bamb_5771 Bamb_5969 NOC: Noc_1891 BPS: BPSL1426 BPSL1535(phbA) BPSL1540 AEH: Mlg_0688 Mlg_2706 BPM: BURPS1710b_2325(bktB) HHA: Hhal_1685 BURPS1710b_2330(phbA) HCH: HCH_05299 BURPS1710b_2453(atoB-2) CSA: Csal_0301 Csal_3068 BPL: BURPS1106A_2197(bktB) ABO: ABO_0648(fadAx) BURPS1106A_2202(phbA) MMW: Mmwyl1_0073 Mmwyl1_3021 Mmwyl1_3053 BPD: BURPS668_2160(bktB) BURPS668_2165(phbA) Mmwyl1_3097 Mmwyl1_4182 BTE: BTH_I2144 BTH_I2256 BTH_I2261 AHA: AHA_2143(atoB) PNU: Pnuc_0927 CVI: CV_2088(atoB) CV_2790(phaA) BPE: BP0447 BP0668 BP2059 RSO: RSc0276(atoB) RSc1632(phbA) RSc1637(bktB) BPA: BPP0608 BPP1744 BPP3805 BPP4216 RSc1761(RS02948) BPP4361 REU: Reut_A0138 Reut_A1348 Reut_A1353 BBR: BB0614 BB3364 BB4250 BB4804 BB4947 Reut_B4561 Reut_B4738 RFR: Rfer_0272 Rfer_1000 Rfer_1871 Rfer_2273 Reut_B5587 Reut_C5943 Reut_C6062 Rfer_2561 Rfer_2594 REH: H16_A0170 H16_A0867 H16_A0868 Rfer_3839 H16_A0872 H16_A1297 POL: Bpro_1577 Bpro_2140 Bpro_3113 Bpro_4187 H16_A1438(phaA) H16_A1445(bktB) H16_A1528 PNA: Pnap_0060 Pnap_0458 Pnap_0867 Pnap_1159 H16_A1713 H16_A1720 Pnap_2136 Pnap_2804 H16_A1887 H16_A2148 H16_B0380 H16_B0381 AAV: Aave_0031 Aave_2478 Aave_3944 Aave_4368 H16_B0406 H16_B0662 AJS: Ajs_0014 Ajs_0124 Ajs_1931 Ajs_2073 H16_B0668 H16_B0759 H16_B1369 H16_B1771 Ajs_2317 Ajs_3548 RME: Rmet_0106 Rmet_1357 Rmet_1362 Rmet_5156 Ajs_3738 Ajs_3776 BMA: BMA1316 BMA1321(phbA) BMA1436 VEI: Veis_1331 Veis_3818 Veis_4193 BMV: BMASAVP1_A1805(bktB) DAC: Daci_0025 Daci_0192 Daci_3601 Daci_5988 BMASAVP1_A1810(phbA) MPT: Mpe_A1536 Mpe_A1776 Mpe_A1869 BML: BMA10299_A0086(phbA) BMA10299_A0091 Mpe_A3367 BMN: BMA10247_1076(bktB) HAR: HEAR0577(phbA) BMA10247_1081(phbA) MMS: mma_0555 NEU: NE2262(bktB) NET: Neut_0610 RPC: RPC_0504 RPC_0636 RPC_0641 RPC_0832 EBA: ebA5202 p2A409(tioL) RPC_1050 RPC_2005 AZO: azo0464(fadA1) azo0469(fadA2) azo2172(thlA) RPC_2194 RPC_2228 DAR: Daro_0098 Daro_3022 RPD: RPD_0306 RPD_0320 RPD_3105 RPD_3306 HPA: HPAG1_0675 RPE: RPE_0168 RPE_0248 RPE_3827 HAC: Hac_0958(atoB) NWI: Nwi_3060 GME: Gmet_1719 Gmet_2074 Gmet_2213 XAU: Xaut_3108 Xaut_4665 Gmet_2268 Gmet_3302 CCR: CC_0510 CC_0894 CC_3462 GUR: Gura_3043 SIL: SPO0142(bktB) SPO0326(phbA) SPO0773 BBA: Bd0404(atoB) Bd2095 SPO3408 DOL: Dole_0671 Dole_1778 Dole_2160 Dole_2187 SIT: TM1040_0067 TM1040_2790 TM1040_3026 ADE: Adeh_0062 Adeh_2365 TM1040_3735 AFW: Anae109_0064 Anae109_1504 RSP: RSP_0745 RSP_1354 RSP_3184 MXA: MXAN_3791 RSH: Rsph17029_0022 Rsph17029_2401 SAT: SYN_02642 Rsph17029_3179 Rsph17029_3921 SFU: Sfum_2280 Sfum_3582 RSQ: Rsph17025_0012 Rsph17025_2466 RPR: RP737 Rsph17025_2833 RCO: RC1134 RC1135 JAN: Jann_0262 Jann_0493 Jann_4050 RFE: RF_0163(paaJ) RDE: RD1_0025 RD1_0201(bktB) RD1_3394(phbA) RBE: RBE_0139(paaJ) PDE: Pden_2026 Pden_2663 Pden_2870 Pden_2907 RAK: A1C_05820 Pden_4811 Pden_5022 RBO: A1I_07215 DSH: Dshi_0074 Dshi_3066 Dshi_3331 RCM: A1E_04760 MMR: Mmar10_0697 PUB: SAR11_0428(thlA) HNE: HNE_2706 HNE_3065 HNE_3133 MLO: mlr3847 NAR: Saro_0809 Saro_1069 Saro_1222 Saro_2306 MES: Meso_3374 Saro_2349 PLA: Plav_1573 Plav_2783 SAL: Sala_0781 Sala_1244 Sala_2896 Sala_3158 SME: SMa1450 SMc03879(phbA) SWI: Swit_0632 Swit_0752 Swit_2893 Swit_3602 SMD: Smed_0499 Smed_3117 Smed_5094 Swit_4887 Swit_5019 Smed_5096 Swit_5309 ATU: Atu2769(atoB) Atu3475 ELI: ELI_01475 ELI_06705 ELI_12035 ATC: AGR_C_5022(phbA) AGR_L_2713 GBE: GbCGDNIH1_0447 RET: RHE_CH04018(phbAch) ACR: Acry_1847 Acry_2256 RHE_PC00068(ypc00040) RHE_PF00014(phbAf) RRU: Rru_A0274 Rru_A1380 Rru_A1469 Rru_A1946 RLE: RL4621(phaA) pRL100301 pRL120369 Rru_A3387 BME: BMEI0274 BMEII0817 MAG: amb0842 BMF: BAB1_1783(phbA-1) BAB2_0790(phbA-2) MGM: Mmc1_1165 BMS: BR1772(phbA-1) BRA0448(phbA-2) ABA: Acid345_3239 BMB: BruAb1_1756(phbA-1) BruAb2_0774(phbA-2) BSU: BG11319(mmgA) BG13063(yhfS) BOV: BOV_1707(phbA-1) BHA: BH1997 BH2029 BH3801(mmgA) OAN: Oant_1130 Oant_3107 Oant_3718 Oant_4020 BAN: BA3687 BA4240 BA5589 BJA: bll0226(atoB) bll3949 bll7400 bll7819 BAR: GBAA3687 GBAA4240 GBAA5589 blr3724(phbA) BAA: BA_0445 BA_4172 BA_4700 BRA: BRADO0562(phbA) BRADO0983(pimB) BAT: BAS3418 BAS3932 BAS5193 BRADO3110 BRADO3134(atoB) BCE: BC3627 BC4023 BC5344 BBT: BBta_3558 BBta_3575(atoB) BBta_5147(pimB) BCA: BCE_3646 BCE_4076 BCE_5475 BBta_7072(pimB) BCZ: BCZK3329(mmgA) BCZK3780(thl) BBta_7614(phbA) BCZK5044(atoB) RPA: RPA0513(pcaF) RPA0531 RPA3715(pimB) BCY: Bcer98_2722 Bcer98_3865 RPB: RPB_0509 RPB_0525 RPB_1748 BTK: BT9727_3379(mmgA) BT9727_3765(thl) BTL: BALH_3262(mmgA) BALH_3642(fadA) BT9727_5028(atoB) BALH_4843(atoB) LJO: LJ1609 BLI: BL03925(mmgA) LAC: LBA0626(thiL) BLD: BLi03968(mmgA) LSA: LSA1486 BCL: ABC0345 ABC2989 ABC3617 LDB: Ldb0879 ABC3891(mmgA) LBU: LBUL_0804 BAY: RBAM_022450 LBR: LVIS_2218 BPU: BPUM_2374(yhfS) BPUM_2941 BPUM_3373 LCA: LSEI_1787 OIH: OB0676 OB0689 OB2632 OB3013 LGA: LGAS_1374 GKA: GK1658 GK3397 LRE: Lreu_0052 SAU: SA0342 SA0534(vraB) EFA: EF1364 SAV: SAV0354 SAV0576(vraB) OOE: OEOE_0529 SAM: MW0330 MW0531(vraB) STH: STH2913 STH725 STH804 SAR: SAR0351(thl) SAR0581 CAC: CAC2873 CA_P0078(thiL) SAS: SAS0330 SAS0534 CPE: CPE2195(atoB) SAC: SACOL0426 SACOL0622(atoB) CPF: CPF_2460 SAB: SAB0304(th1) SAB0526 CPR: CPR_2170 SAA: SAUSA300_0355 SAUSA300_0560(vraB) CTC: CTC00312 SAO: SAOUHSC_00336 SAOUHSC_00558 CNO: NT01CX_0538 NT01CX_0603 SAJ: SaurJH9_0402 CDF: CD1059(thlA1) CD2676(thlA2) SAH: SaurJH1_0412 CBO: CBO3200(thl) SEP: SE0346 SE2384 CBE: Cbei_0411 Cbei_3630 SER: SERP0032 SERP0220 CKL: CKL_3696(thlA1) CKL_3697(thlA2) SHA: SH0510(mvaC) SH2417 CKL_3698(thlA3) SSP: SSP0325 SSP2145 AMT: Amet_4630 LMO: lmo1414 AOE: Clos_0084 Clos_0258 LMF: LMOf2365_1433 CHY: CHY_1288 CHY_1355(atoB) CHY_1604 LIN: lin1453 CHY_1738 LWE: lwe1431 DSY: DSY0632 DSY0639 DSY1567 DSY1710 LLA: L11745(thiL) L25946(fadA) DSY2402 DSY3302 LLC: LACR_1665 LACR_1956 DRM: Dred_0400 Dred_1491 Dred_1784 Dred_1892 LLM: llmg_0930(thiL) SWO: Swol_0308 Swol_0675 Swol_0789 Swol_1486 SPY: SPy_0140 SPy_1637(atoB) Swol_1934 Swol_2051 SPZ: M5005_Spy_0119 M5005_Spy_0432 TTE: TTE0549(paaJ) M5005_Spy_1344(atoB) MTA: Moth_1260 SPM: spyM18_0136 spyM18_1645(atoB) MTU: Rv1135A Rv1323(fadA4) Rv3546(fadA5) SPG: SpyM3_0108 SpyM3_1378(atoB) MTC: MT1365(phbA) SPS: SPs0110 SPs0484 MBO: Mb1167 Mb1358(fadA4) Mb3576(fadA5) SPH: MGAS10270_Spy0121 MGAS10270_Spy0433 Mb3586c(fadA6) MGAS10270_Spy1461(atoB) MBB: BCG_1197 BCG_1385(fadA4) SPI: MGAS10750_Spy0124 MGAS10750_Spy0452 BCG_3610(fadA5) BCG_3620c(fadA6) MGAS10750_Spy1453(atoB) MLE: ML1158(fadA4) SPJ: MGAS2096_Spy0123 MGAS2096_Spy0451 MPA: MAP2407c(fadA3) MAP2436c(fadA4) MGAS2096_Spy1365(atoB) MAV: MAV_1544 MAV_1573 MAV_1863 SPK: MGAS9429_Spy0121 MGAS9429_Spy0431 MAV_5081 MGAS9429_Spy1339(atoB) MSM: MSMEG_2224 MSMEG_4920 SPF: SpyM50447(atoB2) MUL: MUL_0357 SPA: M6_Spy0166 M6_Spy0466 M6_Spy1390 MVA: Mvan_1976 Mvan_1988 Mvan_4305 SPB: M28_Spy0117 M28_Spy0420 Mvan_4677 Mvan_4891 M28_Spy1385(atoB) MGI: Mflv_1347 Mflv_1484 Mflv_2040 Mflv_2340 SAK: SAK_0568 Mflv_4356 Mflv_4368 MMC: Mmcs_1758 Mmcs_1769 Mmcs_3796 FJO: Fjoh_4612 Mmcs_3864 FPS: FP0770 FP1586 FP1725 MKM: Mkms_0251 Mkms_1540 Mkms_1805 RRS: RoseRS_3911 RoseRS_4348 Mkms_1816 Mkms_2836 Mkms_3159 RCA: Rcas_0702 Rcas_3206 Mkms_3286 Mkms_3869 Mkms_3938 Mkms_4227 HAU: Haur_0522 Mkms_4411 Mkms_4580 DRA: DR_1072 DR_1428 DR_1960 DR_2480 Mkms_4724 Mkms_4764 Mkms_4776 DR_A0053 MJL: Mjls_0231 Mjls_1739 Mjls_1750 Mjls_2819 DGE: Dgeo_0755 Dgeo_1305 Dgeo_1441 Dgeo_1883 Mjls_3119 Mjls_3235 TTH: TTC0191 TTC0330 Mjls_3800 Mjls_3850 Mjls_4110 Mjls_4383 TTJ: TTHA0559 Mjls_4705 Mjls_4876 TME: Tmel_1134 Mjls_5018 Mjls_5063 Mjls_5075 FNO: Fnod_0314 CGL: NCgl2309(cgl2392) PMO: Pmob_0515 CGB: cg2625(pcaF) HMA: rrnAC0896(acaB3) rrnAC2815(aca2) CEF: CE0731 CE2295 rrnAC3497(yqeF) CJK: jk1543(fadA3) rrnB0240(aca1) rrnB0242(acaB2) rrnB0309(acaB1) NFA: nfa10750(fadA4) TAC: Ta0582 RHA: RHA1_ro01455 RHA1_ro01623 TVO: TVN0649 RHA1_ro01876 RHA1_ro02517(catF) PTO: PTO1505 RHA1_ro03022 RHA1_ro03024 RHA1_ro03391 APE: APE_2108 RHA1_ro03892 SSO: SSO2377(acaB-4) RHA1_ro04599 RHA1_ro05257 RHA1_ro08871 STO: ST0514 SCO: SCO5399(SC8F4.03) SAI: Saci_0963 Saci_1361(acaB1) SMA: SAV1384(fadA5) SAV2856(fadA1) MSE: Msed_0656 ART: Arth_1160 Arth_2986 Arth_3268 Arth_4073 PAI: PAE1220 NCA: Noca_1371 Noca_1797 Noca_1828 Noca_2764 PIS: Pisl_0029 Pisl_1301 Noca_4142 PCL: Pcal_0781 TFU: Tfu_1520 Tfu_2394 PAS: Pars_0309 Pars_1071 FRA: Francci3_3687 CMA: Cmaq_1941 FRE: Franean1_1044 Franean1_2711 Franean1_2726 Franean1_3929 Franean1_4037 Franean1_4577 FAL: FRAAL2514 FRAAL2618 FRAAL5910(atoB) ACE: Acel_0626 Acel_0672 SEN: SACE_1192(mmgA) SACE_2736(fadA6) SACE_4011(catF) SACE_6236(fadA4) STP: Strop_3610 SAQ: Sare_1316 Sare_3991 RXY: Rxyl_1582 Rxyl_1842 Rxyl_2389 Rxyl_2530 FNU: FN0495 BGA: BG0110(fadA) BAF: BAPKO_0110(fadA) LIL: LA0457(thiL1) LA0828(thiL2) LA4139(fadA) LIC: LIC10396(phbA) LBJ: LBJ_2862(paaJ-4) LBL: LBL_0209(paaJ-4) SYN: slr1993(phaA) SRU: SRU_1211(atoB) SRU_1547 CHU: CHU_1910(atoB) GFO: GFO_1507(atoB) Exemplary HMG-CoA synthase nucleic acids and polypeptides HSA: 3157(HMGCS1) 3158(HMGCS2) MXA: MXAN_3948(tac) MXAN_4267(mvaS) PTR: 457169(HMGCS2) 461892(HMGCS1) BSU: BG10926(pksG) MCC: 702553(HMGCS1) 713541(HMGCS2) OIH: OB2248 MMU: 15360(Hmgcs2) 208715(Hmgcs1) SAU: SA2334(mvaS) RNO: 24450(Hmgcs2) 29637(Hmgcs1) SAV: SAV2546(mvaS) CFA: 479344(HMGCS1) 607923(HMGCS2) SAM: MW2467(mvaS) BTA: 407767(HMGCS1) SAR: SAR2626(mvaS) SSC: 397673(CH242-38B5.1) SAS: SAS2432 GGA: 396379(HMGCS1) SAC: SACOL2561 XLA: 380091(hmgcs1) 447204(MGC80816) SAB: SAB2420(mvaS) DRE: 394060(hmgcs1) SAA: SAUSA300_2484 SPU: 578259(LOC578259) SAO: SAOUHSC_02860 DME: Dmel_CG4311(Hmgs) SAJ: SaurJH9_2569 CEL: F25B4.6 SAH: SaurJH1_2622 ATH: AT4G11820(BAP1) SEP: SE2110 OSA: 4331418 4347614 SER: SERP2122 CME: CMM189C SHA: SH0508(mvaS) SCE: YML126C(ERG13) SSP: SSP0324 AGO: AGOS_ADL356C LMO: lmo1415 PIC: PICST_83020 LMF: LMOf2365_1434(mvaS) CAL: CaO19_7312(CaO19.7312) LIN: lin1454 CGR: CAGL0H04081g LWE: lwe1432(mvaS) SPO: SPAC4F8.14c(hcs) LLA: L13187(hmcM) MGR: MGG_01026 LLC: LACR_1666 ANI: AN4923.2 LLM: llmg_0929(hmcM) AFM: AFUA_3G10660 AFUA_8G07210 SPY: SPy_0881(mvaS.2) AOR: AO090003000611 AO090010000487 SPZ: M5005_Spy_0687(mvaS.1) CNE: CNC05080 CNG02670 SPM: spyM18_0942(mvaS2) UMA: UM05362.1 SPG: SpyM3_0600(mvaS.2) ECU: ECU10_0510 SPS: SPs1253 DDI: DDBDRAFT_0217522 DDB_0219924(hgsA) SPH: MGAS10270_Spy0745(mvaS1) TET: TTHERM_00691190 SPI: MGAS10750_Spy0779(mvaS1) TBR: Tb927.8.6110 SPJ: MGAS2096_Spy0759(mvaS1) YPE: YPO1457 SPK: MGAS9429_Spy0743(mvaS1) YPK: y2712(pksG) SPF: SpyM51121(mvaS) YPM: YP_1349(pksG) SPA: M6_Spy0704 YPA: YPA_0750 SPB: M28_Spy0667(mvaS.1) YPN: YPN_2521 SPN: SP_1727 YPP: YPDSF_1517 SPR: spr1571(mvaS) YPS: YPTB1475 SPD: SPD_1537(mvaS) CBD: COXBU7E912_1931 SAG: SAG1316 TCX: Tcr_1719 SAN: gbs1386 DNO: DNO_0799 SAK: SAK_1347 BMA: BMAA1212 SMU: SMU.943c BPS: BPSS1002 STC: str0577(mvaS) BPM: BURPS1710b_A2613 STL: stu0577(mvaS) BPL: BURPS1106A_A1384 STE: STER_0621 BPD: BURPS668_A1470 SSA: SSA_0338(mvaS) BTE: BTH_II1670 SSU: SSU05_1641 SSV: SSU98_1652 OOE: OEOE_0968 SGO: SGO_0244 LME: LEUM_1184 LPL: lp_2067(mvaS) NFA: nfa22120 LJO: LJ1607 SEN: SACE_4570(pksG) LAC: LBA0628(hmcS) BBU: BB0683 LSA: LSA1484(mvaS) BGA: BG0706 LSL: LSL_0526 BAF: BAPKO_0727 LDB: Ldb0881(mvaS) FJO: Fjoh_0678 LBU: LBUL_0806 HAL: VNG1615G(mvaB) LBR: LVIS_1363 HMA: rrnAC1740(mvaS) LCA: LSEI_1785 HWA: HQ2868A(mvaB) LGA: LGAS_1372 NPH: NP2608A(mvaB_1) NP4836A(mvaB_2) LRE: Lreu_0676 PPE: PEPE_0868 EFA: EF1363 Exemplary hydroxymethylglutaryl-CoA reductase nucleic acids and polypeptides HSA: 3156(HMGCR) PAT: Patl_0427 PTR: 471516(HMGCR) CBU: CBU_0030 CBU_0610 MCC: 705479(HMGCR) CBD: COXBU7E912_0151 MMU: 15357(Hmgcr) COXBU7E912_0622(hmgA) RNO: 25675(Hmgcr) TCX: Tcr_1717 CFA: 479182(HMGCR) DNO: DNO_0797 BTA: 407159(HMGCR) CVI: CV_1806 GGA: 395145(RCJMB04_14m24) SUS: Acid_5728 Acid_6132 SPU: 373355(LOC373355) SAU: SA2333(mvaA) DME: Dmel_CG10367(Hmgcr) SAV: SAV2545(mvaA) CEL: F08F8.2 SAM: MW2466(mvaA) OSA: 4347443 SAB: SAB2419c(mvaA) SCE: YLR450W(HMG2) YML075C(HMG1) SEP: SE2109 AGO: AGOS_AER152W LWE: lwe0819(mvaA) CGR: CAGL0L11506g LLA: L10433(mvaA) SPO: SPCC162.09c(hmg1) LLC: LACR_1664 ANI: AN3817.2 LLM: llmg_0931(mvaA) AFM: AFUA_1G11230 AFUA_2G03700 SPY: SPy_0880(mvaS.1) AOR: AO090103000311 AO090120000217 SPM: spyM18_0941(mvaS1) CNE: CNF04830 SPG: SpyM3_0599(mvaS.1) UMA: UM03014.1 SPS: SPs1254 ECU: ECU10_1720 SPH: MGAS10270_Spy0744 DDI: DDB_0191125(hmgA) DDB_0215357(hmgB) SPI: MGAS10750_Spy0778 TBR: Tb927.6.4540 SPJ: MGAS2096_Spy0758 TCR: 506831.40 509167.20 SPK: MGAS9429_Spy0742 LMA: LmjF30.3190 SPA: M6_Spy0703 VCH: VCA0723 SPN: SP_1726 VCO: VC0395_0662 SAG: SAG1317 VVU: VV2_0117 SAN: gbs1387 VVY: VVA0625 STC: str0576(mvaA) VPA: VPA0968 STL: stu0576(mvaA) VFI: VFA0841 STE: STER_0620 SSA: SSA_0337(mvaA) HAL: VNG1875G(mvaA) LPL: lp_0447(mvaA) HMA: rrnAC3412(mvaA) LJO: LJ1608 HWA: HQ3215A(hmgR) LSL: LSL_0224 NPH: NP0368A(mvaA_2) NP2422A(mvaA_1) LBR: LVIS_0450 TAC: Ta0406m LGA: LGAS_1373 TVO: TVN1168 EFA: EF1364 PTO: PTO1143 NFA: nfa22110 PAB: PAB2106(mvaA) BGA: BG0708(mvaA) PFU: PF1848 SRU: SRU_2422 TKO: TK0914 FPS: FP2341 RCI: RCIX1027(hmgA) RCIX376(hmgA) MMP: MMP0087(hmgA) APE: APE_1869 MMQ: MmarC5_1589 IHO: Igni_0476 MAC: MA3073(hmgA) HBU: Hbut_1531 MBA: Mbar_A1972 SSO: SSO0531 MMA: MM_0335 STO: ST1352 MBU: Mbur_1098 SAI: Saci_1359 MHU: Mhun_3004 PAI: PAE2182 MEM: Memar_2365 PIS: Pisl_0814 MBN: Mboo_0137 PCL: Pcal_1085 MTH: MTH562 PAS: Pars_0796 MST: Msp_0584(hmgA) MSI: Msm_0227 MKA: MK0355(HMG1) AFU: AF1736(mvaA) Exemplary mevalonate kinase nucleic acids and polypeptides HSA: 4598(MVK) TET: TTHERM_00637680 MCC: 707645(MVK) TBR: Tb927.4.4070 MMU: 17855(Mvk) TCR: 436521.9 509237.10 RNO: 81727(Mvk) LMA: LmjF31.0560 CFA: 486309(MVK) CBU: CBU_0608 CBU_0609 BTA: 505792(MVK) CBD: COXBU7E912_0620(mvk) GGA: 768555(MVK) LPN: lpg2039 DRE: 492477(zgc: 103473) LPF: lpl2017 SPU: 585785(LOC585785) LPP: lpp2022 DME: Dmel_CG33671 BBA: Bd1027(lmbP) Bd1630(mvk) OSA: 4348331 MXA: MXAN_5019(mvk) SCE: YMR208W(ERG12) OIH: OB0225 AGO: AGOS_AER335W SAU: SA0547(mvaK1) PIC: PICST_40742(ERG12) SAV: SAV0590(mvaK1) CGR: CAGL0F03861g SAM: MW0545(mvaK1) SPO: SPAC13G6.11c SAR: SAR0596(mvaK1) MGR: MGG_06946 SAS: SAS0549 ANI: AN3869.2 SAC: SACOL0636(mvk) AFM: AFUA_4G07780 SAB: SAB0540(mvaK1) AOR: AO090023000793 SAA: SAUSA300_0572(mvk) CNE: CNK01740 SAO: SAOUHSC_00577 ECU: ECU09_1780 SEP: SE0361 DDI: DDBDRAFT_0168621 SER: SERP0238(mvk) SHA: SH2402(mvaK1) LCA: LSEI_1491 SSP: SSP2122 LGA: LGAS_1033 LMO: lmo0010 LRE: Lreu_0915 LMF: LMOf2365_0011 PPE: PEPE_0927 LIN: lin0010 EFA: EF0904(mvk) LWE: lwe0011(mvk) OOE: OEOE_1100 LLA: L7866(yeaG) LME: LEUM_1385 LLC: LACR_0454 NFA: nfa22070 LLM: llmg_0425(mvk) BGA: BG0711 SPY: SPy_0876(mvaK1) BAF: BAPKO_0732 SPZ: M5005_Spy_0682(mvaK1) FPS: FP0313 SPM: spyM18_0937(mvaK1) MMP: MMP1335 SPG: SpyM3_0595(mvaK1) MAE: Maeo_0775 SPS: SPs1258 MAC: MA0602(mvk) SPH: MGAS10270_Spy0740(mvaK1) MBA: Mbar_A1421 SPI: MGAS10750_Spy0774(mvaK1) MMA: MM_1762 SPJ: MGAS2096_Spy0753(mvaK1) MBU: Mbur_2395 SPK: MGAS9429_Spy0737(mvaK1) MHU: Mhun_2890 SPF: SpyM51126(mvaK1) MEM: Memar_1812 SPA: M6_Spy0699 MBN: Mboo_2213 SPB: M28_Spy0662(mvaK1) MST: Msp_0858(mvk) SPN: SP_0381 MSI: Msm_1439 SPR: spr0338(mvk) MKA: MK0993(ERG12) SPD: SPD_0346(mvk) HAL: VNG1145G(mvk) SAG: SAG1326 HMA: rrnAC0077(mvk) SAN: gbs1396 HWA: HQ2925A(mvk) SAK: SAK_1357(mvk) NPH: NP2850A(mvk) SMU: SMU.181 PTO: PTO1352 STC: str0559(mvaK1) PHO: PH1625 STL: stu0559(mvaK1) PAB: PAB0372(mvk) STE: STER_0598 PFU: PF1637(mvk) SSA: SSA_0333(mvaK1) TKO: TK1474 SSU: SSU05_0289 RCI: LRC399(mvk) SSV: SSU98_0285 APE: APE_2439 SGO: SGO_0239(mvk) HBU: Hbut_0877 LPL: lp_1735(mvaK1) SSO: SSO0383 LJO: LJ1205 STO: ST2185 LAC: LBA1167(mvaK) SAI: Saci_2365(mvk) LSA: LSA0908(mvaK1) MSE: Msed_1602 LSL: LSL_0685(eRG) PAI: PAE3108 LDB: Ldb0999(mvk) PIS: Pisl_0467 LBU: LBUL_0906 PCL: Pcal_1835 LBR: LVIS_0858 Exemplary phosphomevalonate kinase nucleic acids and polypeptides HSA: 10654(PMVK) CFA: 612251(PMVK) PTR: 457350(PMVK) BTA: 513533(PMVK) MCC: 717014(PMVK) DME: Dmel_CG10268 MMU: 68603(Pmvk) ATH: AT1G31910 OSA: 4332275 SPS: SPs1256 SCE: YMR220W(ERG8) SPH: MGAS10270_Spy0742(mvaK2) AGO: AGOS_AER354W SPI: MGAS10750_Spy0776(mvaK2) PIC: PICST_52257(ERG8) SPJ: MGAS2096_Spy0755(mvaK2) CGR: CAGL0F03993g SPK: MGAS9429_Spy0739(mvaK2) SPO: SPAC343.01c SPF: SpyM51124(mvaK2) MGR: MGG_05812 SPA: M6_Spy0701 ANI: AN2311.2 SPB: M28_Spy0664(mvaK2) AFM: AFUA_5G10680 SPN: SP_0383 AOR: AO090010000471 SPR: spr0340(mvaK2) CNE: CNM00100 SPD: SPD_0348(mvaK2) UMA: UM00760.1 SAG: SAG1324 DDI: DDBDRAFT_0184512 SAN: gbs1394 TBR: Tb09.160.3690 SAK: SAK_1355 TCR: 507913.20 508277.140 SMU: SMU.938 LMA: LmjF15.1460 STC: str0561(mvaK2) MXA: MXAN_5017 STL: stu0561(mvaK2) OIH: OB0227 STE: STER_0600 SAU: SA0549(mvaK2) SSA: SSA_0335(mvaK2) SAV: SAV0592(mvaK2) SSU: SSU05_0291 SAM: MW0547(mvaK2) SSV: SSU98_0287 SAR: SAR0598(mvaK2) SGO: SGO_0241 SAS: SAS0551 LPL: lp_1733(mvaK2) SAC: SACOL0638 LJO: LJ1207 SAB: SAB0542(mvaK2) LAC: LBA1169 SAA: SAUSA300_0574 LSA: LSA0906(mvaK2) SAO: SAOUHSC_00579 LSL: LSL_0683 SAJ: SaurJH9_0615 LDB: Ldb0997(mvaK) SEP: SE0363 LBU: LBUL_0904 SER: SERP0240 LBR: LVIS_0860 SHA: SH2400(mvaK2) LCA: LSEI_1092 SSP: SSP2120 LGA: LGAS_1035 LMO: lmo0012 LRE: Lreu_0913 LMF: LMOf2365_0013 PPE: PEPE_0925 LIN: lin0012 EFA: EF0902 LWE: lwe0013 NFA: nfa22090 LLA: L10014(yebA) BGA: BG0710 LLC: LACR_0456 BAF: BAPKO_0731 LLM: llmg_0427 NPH: NP2852A SPY: SPy_0878(mvaK2) SSO: SSO2988 SPZ: M5005_Spy_0684(mvaK2) STO: ST0978 SPM: spyM18_0939 SAI: Saci_1244 SPG: SpyM3_0597(mvaK2) Exemplary diphosphomevalonate decarboxylase nucleic acids and polypeptides HSA: 4597(MVD) CFA: 489663(MVD) PTR: 468069(MVD) GGA: 425359(MVD) MCC: 696865(MVD) DME: Dmel_CG8239 MMU: 192156(Mvd) SCE: YNR043W(MVD1) RNO: 81726(Mvd) AGO: AGOS_AGL232C PIC: PICST_90752 SPH: MGAS10270_Spy0741(mvaD) CGR: CAGL0C03630g SPI: MGAS10750_Spy0775(mvaD) SPO: SPAC24C9.03 SPJ: MGAS2096_Spy0754(mvaD) MGR: MGG_09750 SPK: MGAS9429_Spy0738(mvaD) ANI: AN4414.2 SPF: SpyM51125(mvaD) AFM: AFUA_4G07130 SPA: M6_Spy0700 AOR: AO090023000862 SPB: M28_Spy0663(mvaD) CNE: CNL04950 SPN: SP_0382 UMA: UM05179.1 SPR: spr0339(mvd1) DDI: DDBDRAFT_0218058 SPD: SPD_0347(mvaD) TET: TTHERM_00849200 SAG: SAG1325(mvaD) TBR: Tb10.05.0010 Tb10.61.2745 SAN: gbs1395 TCR: 507993.330 511281.40 SAK: SAK_1356(mvaD) LMA: LmjF18.0020 SMU: SMU.937 CBU: CBU_0607(mvaD) STC: str0560(mvaD) CBD: COXBU7E912_0619(mvaD) STL: stu0560(mvaD) LPN: lpg2040 STE: STER_0599 LPF: lpl2018 SSA: SSA_0334(mvaD) LPP: lpp2023 SSU: SSU05_0290 TCX: Tcr_1734 SSV: SSU98_0286 DNO: DNO_0504(mvaD) SGO: SGO_0240(mvaD) BBA: Bd1629 LPL: lp_1734(mvaD) MXA: MXAN_5018(mvaD) LJO: LJ1206 OIH: OB0226 LAC: LBA1168(mvaD) SAU: SA0548(mvaD) LSA: LSA0907(mvaD) SAV: SAV0591(mvaD) LSL: LSL_0684 SAM: MW0546(mvaD) LDB: Ldb0998(mvaD) SAR: SAR0597(mvaD) LBU: LBUL_0905 SAS: SAS0550 LBR: LVIS_0859 SAC: SACOL0637(mvaD) LCA: LSEI_1492 SAB: SAB0541(mvaD) LGA: LGAS_1034 SAA: SAUSA300_0573(mvaD) LRE: Lreu_0914 SAO: SAOUHSC_00578 PPE: PEPE_0926 SAJ: SaurJH9_0614 EFA: EF0903(mvaD) SAH: SaurJH1_0629 LME: LEUM_1386 SEP: SE0362 NFA: nfa22080 SER: SERP0239(mvaD) BBU: BB0686 SHA: SH2401(mvaD) BGA: BG0709 SSP: SSP2121 BAF: BAPKO_0730 LMO: lmo0011 GFO: GFO_3632 LMF: LMOf2365_0012(mvaD) FPS: FP0310(mvaD) LIN: lin0011 HAU: Haur_1612 LWE: lwe0012(mvaD) HAL: VNG0593G(dmd) LLA: L9089(yeaH) HMA: rrnAC1489(dmd) LLC: LACR_0455 HWA: HQ1525A(mvaD) LLM: llmg_0426(mvaD) NPH: NP1580A(mvaD) SPY: SPy_0877(mvaD) PTO: PTO0478 PTO1356 SPZ: M5005_Spy_0683(mvaD) SSO: SSO2989 SPM: spyM18_0938(mvd) STO: ST0977 SPG: SpyM3_0596(mvaD) SAI: Saci_1245(mvd) SPS: SPs1257 MSE: Msed_1576 Exemplary isopentenyl phosphate kinases (IPK) nucleic acids and polypeptides Methanobacterium thermoautotrophicum Picrophilus torridus DSM9790 (IG-57) gi|48477569 gi|2621082 Pyrococcus abyssi gi|14520758 Methanococcus jannaschii DSM 2661 gi|1590842; Pyrococcus horikoshii OT3 gi|3258052 Methanocaldococcus jannaschii gi|1590842 Archaeoglobus fulgidus DSM4304 gi|2648231 Methanothermobacter thermautotrophicus gi|2621082 Exemplary isopentenyl-diphosphate Delta-isomerase (IDI) nucleic acids and polypeptides HSA: 3422(IDI1) 91734(IDI2) STY: STY3195 PTR: 450262(IDI2) 450263(IDI1) STT: t2957 MCC: 710052(LOC710052) 721730(LOC721730) SPT: SPA2907(idi) MMU: 319554(Idi1) SEC: SC2979(idi) RNO: 89784(Idi1) STM: STM3039(idi) GGA: 420459(IDI1) SFL: SF2875(idi) XLA: 494671(LOC494671) SFX: S3074 XTR: 496783(idi2) SFV: SFV_2937 SPU: 586184(LOC586184) SSN: SSON_3042 SSON_3489(yhfK) CEL: K06H7.9(idi-1) SBO: SBO_3103 ATH: AT3G02780(IPP2) SDY: SDY_3193 OSA: 4338791 4343523 ECA: ECA2789 CME: CMB062C PLU: plu3987 SCE: YPL117C(IDI1) ENT: Ent638_3307 AGO: AGOS_ADL268C SPE: Spro_2201 PIC: PICST_68990(IDI1) VPA: VPA0278 CGR: CAGL0J06952g VFI: VF0403 SPO: SPBC106.15(idi1) PPR: PBPRA0469(mvaD) ANI: AN0579.2 PEN: PSEEN4850 AFM: AFUA_6G11160 CBU: CBU_0607(mvaD) AOR: AO090023000500 CBD: COXBU7E912_0619(mvaD) CNE: CNA02550 LPN: lpg2051 UMA: UM04838.1 LPF: lpl2029 ECU: ECU02_0230 LPP: lpp2034 DDI: DDB_0191342(ipi) TCX: Tcr_1718 TET: TTHERM_00237280 TTHERM_00438860 HHA: Hhal_1623 TBR: Tb09.211.0700 DNO: DNO_0798 TCR: 408799.19 510431.10 EBA: ebA5678 p2A143 LMA: LmjF35.5330 DVU: DVU1679(idi) EHI: 46.t00025 DDE: Dde_1991 ECO: b2889(idi) LIP: LI1134 ECJ: JW2857(idi) BBA: Bd1626 ECE: Z4227 AFW: Anae109_4082 ECS: ECs3761 MXA: MXAN_5021(fni) ECC: c3467 RPR: RP452 ECI: UTI89_C3274 RTY: RT0439(idi) ECP: ECP_2882 RCO: RC0744 ECV: APECO1_3638 RFE: RF_0785(fni) ECW: EcE24377A_3215(idi) RBE: RBE_0731(fni) ECX: EcHS_A3048 RAK: A1C_04190 RBO: A1I_04755 SPZ: M5005_Spy_0685 RCM: A1E_02555 SPM: spyM18_0940 RRI: A1G_04195 SPG: SpyM3_0598 MLO: mlr6371 SPS: SPs1255 RET: RHE_PD00245(ypd00046) SPH: MGAS10270_Spy0743 XAU: Xaut_4134 SPI: MGAS10750_Spy0777 SIL: SPO0131 SPJ: MGAS2096_Spy0756 SIT: TM1040_3442 SPK: MGAS9429_Spy0740 RSP: RSP_0276 SPF: SpyM51123(fni) RSH: Rsph17029_1919 SPA: M6_Spy0702 RSQ: Rsph17025_1019 SPB: M28_Spy0665 JAN: Jann_0168 SPN: SP_0384 RDE: RD1_0147(idi) SPR: spr0341(fni) DSH: Dshi_3527 SPD: SPD_0349(fni) BSU: BG11440(ypgA) SAG: SAG1323 BAN: BA1520 SAN: gbs1393 BAR: GBAA1520 SAK: SAK_1354(fni) BAA: BA_2041 SMU: SMU.939 BAT: BAS1409 STC: str0562(idi) BCE: BC1499 STL: stu0562(idi) BCA: BCE_1626 STE: STER_0601 BCZ: BCZK1380(fni) SSA: SSA_0336 BCY: Bcer98_1222 SGO: SGO_0242 BTK: BT9727_1381(fni) LPL: lp_1732(idi1) BTL: BALH_1354 LJO: LJ1208 BLI: BL02217(fni) LAC: LBA1171 BLD: BLi02426 LSA: LSA0905(idi) BAY: RBAM_021020(fni) LSL: LSL_0682 BPU: BPUM_2020(fni) LDB: Ldb0996(fni) OIH: OB0537 LBU: LBUL_0903 SAU: SA2136(fni) LBR: LVIS_0861 SAV: SAV2346(fni) LCA: LSEI_1493 SAM: MW2267(fni) LGA: LGAS_1036 SAR: SAR2431(fni) LRE: Lreu_0912 SAS: SAS2237 EFA: EF0901 SAC: SACOL2341(fni) OOE: OEOE_1103 SAB: SAB2225c(fni) STH: STH1674 SAA: SAUSA300_2292(fni) CBE: Cbei_3081 SAO: SAOUHSC_02623 DRM: Dred_0474 SEP: SE1925 SWO: Swol_1341 SER: SERP1937(fni-2) MTA: Moth_1328 SHA: SH0712(fni) MTU: Rv1745c(idi) SSP: SSP0556 MTC: MT1787(idi) LMO: lmo1383 MBO: Mb1774c(idi) LMF: LMOf2365_1402(fni) MBB: BCG_1784c(idi) LIN: lin1420 MPA: MAP3079c LWE: lwe1399(fni) MAV: MAV_3894(fni) LLA: L11083(yebB) MSM: MSMEG_1057(fni) MSMEG_2337(fni) LLC: LACR_0457 MUL: MUL_0380(idi2) LLM: llmg_0428(fni) MVA: Mvan_1582 Mvan_2176 SPY: SPy_0879 MGI: Mflv_1842 Mflv_4187 MMC: Mmcs_1954 TTJ: TTHB110 MKM: Mkms_2000 MJA: MJ0862 MIL: Mjls_1934 MMP: MMP0043 CGL: NCgl2223(cgl2305) MMQ: MmarC5_1637 CGB: cg2531(idi) MMX: MmarC6_0906 CEF: CE2207 MMZ: MmarC7_1040 CDI: DIP1730(idi) MAE: Maeo_1184 NFA: nfa19790 nfa22100 MVN: Mevan_1058 RHA: RHA1_ro00239 MAC: MA0604(idi) SCO: SCO6750(SC5F2A.33c) MBA: Mbar_A1419 SMA: SAV1663(idi) MMA: MM_1764 LXX: Lxx23810(idi) MBU: Mbur_2397 CMI: CMM_2889(idiA) MTP: Mthe_0474 AAU: AAur_0321(idi) MHU: Mhun_2888 PAC: PPA2115 MLA: Mlab_1665 FRA: Francci3_4188 MEM: Memar_1814 FRE: Franean1_5570 MBN: Mboo_2211 FAL: FRAAL6504(idi) MTH: MTH48 KRA: Krad_3991 MST: Msp_0856(fni) SEN: SACE_2627(idiB_2) SACE_5210(idi) MSI: Msm_1441 STP: Strop_4438 MKA: MK0776(lldD) SAQ: Sare_4564 Sare_4928 AFU: AF2287 RXY: Rxyl_0400 HAL: VNG1818G(idi) VNG6081G(crt_1) BBU: BB0684 VNG6445G(crt_2) VNG7060 VNG7149 BGA: BG0707 HMA: rrnAC3484(idi) SYN: sll1556 HWA: HQ2772A(idiA) HQ2847A(idiB) SYC: syc2161_c NPH: NP0360A(idiB_1) NP4826A(idiA) SYF: Synpcc7942_1933 NP5124A(idiB_2) CYA: CYA_2395(fni) TAC: Ta0102 CYB: CYB_2691(fni) TVO: TVN0179 TEL: tll1403 PTO: PTO0496 ANA: all4591 PHO: PH1202 AVA: Ava_2461 Ava_B0346 PAB: PAB1662 TER: Tery_1589 PFU: PF0856 SRU: SRU_1900(idi) TKO: TK1470 CHU: CHU_0674(idi) RCI: LRC397(fni) GFO: GFO_2363(idi) APE: APE_1765.1 FJO: Fjoh_0269 SMR: Smar_0822 FPS: FP1792(idi) IHO: Igni_0804 CTE: CT0257 HBU: Hbut_0539 CCH: Cag_1445 SSO: SSO0063 CPH: Cpha266_0385 STO: ST2059 PVI: Cvib_1545 SAI: Saci_0091 PLT: Plut_1764 MSE: Msed_2136 RRS: RoseRS_2437 PAI: PAE0801 RCA: Rcas_2215 PIS: Pisl_1093 HAU: Haur_4687 PCL: Pcal_0017 DRA: DR_1087 PAS: Pars_0051 DGE: Dgeo_1381 TPE: Tpen_0272 TTH: TT_P0067 Exemplary isoprene synthase nucleic acids and polypeptides Genbank Accession Nos. AY341431 AY316691 AY279379 AJ457070  AY182241 

The invention claimed is:
 1. A method for producing a fuel constituent from a bioisoprene composition comprising chemically transforming isoprene in the bioisoprene composition to non-isoprene compounds by: (a) subjecting the bioisoprene composition to heat or catalytic conditions suitable for isoprene dimerization to produce an isoprene dimer and then catalytically hydrogenating the isoprene dimer to form a saturated C10 fuel constituent; or (b) (i) partially hydrogenating the bioisoprene composition to produce an isoamylene, (ii) dimerizing the isoamylene with a mono-olefin selected from the group consisting of isoamylene, propylene and isobutene to form a dimate and (iii) completely hydrogenating the dimate to produce a fuel constituent; wherein the bioisoprene composition is produced by biological means.
 2. The method of claim 1, wherein at least about 95% of isoprene in the bioisoprene composition is converted to non-isoprene compounds.
 3. The method of claim 1, wherein the bioisoprene composition is heated to about 150° C. to about 250° C. to produce an unsaturated cyclic isoprene dimer and the unsaturated cyclic isoprene dimer is hydrogenated catalytically to produce a saturated cyclic isoprene dimer fuel constituent.
 4. The method of claim 1, wherein the method comprises: (i) contacting the bioisoprene composition with a catalyst for catalyzing cyclo-dimerization of isoprene to produce an unsaturated cyclic isoprene dimer and the unsaturated cyclic isoprene dimer is hydrogenated catalytically to produce a saturated cyclic isoprene dimer fuel constituent.
 5. The method of claim 4, wherein the catalyst for catalyzing cyclo-dimerization of isoprene comprising a catalyst selected from the group consisting of a nickel catalyst, iron catalysts and chromium catalysts.
 6. The method of claim 1, wherein the step of partially hydrogenating the bioisoprene composition comprises contacting the bioisoprene composition with hydrogen gas and a catalyst for catalyzing partial hydrogenation of isoprene.
 7. The method of claim 6, wherein the catalyst for catalyzing partial hydrogenation of isoprene comprises a palladium catalyst.
 8. The method of claim 1, wherein the step of dimerizing the isoamylene with a mono-olefin comprises contacting the isoamylene with the mono-olefin in the presence of a catalyst for catalyzing dimerization of mono-olefin.
 9. The method of claim 8, wherein the catalyst for catalyzing dimerization of mono-olefin comprises an acid catalyst.
 10. The method of claim 1, further comprising purifying the isoprene from the bioisoprene composition prior to chemically transforming the bioisoprene composition to a fuel constituent.
 11. A system for producing a fuel constituent from a bioisoprene composition, wherein isoprene in the bioisoprene composition is chemically converted to non-isoprene compounds, the system comprising a bioisoprene composition and: (a) (i) one or more chemicals capable of dimerizing isoprene in the bioisoprene composition or a source of heat capable of dimerizing isoprene in the bioisoprene composition; and (ii) a catalyst capable of hydrogenating the isoprene dimer to form a saturated C10 fuel constituent; or (b) (i) a chemical capable of partially hydrogenating isoprene in the bioisoprene composition to produce an isoamylene, (ii) a chemical capable of dimerizing the isoamylene with mono-olefins selected from the group consisting of isoamylene, propylene and isobutene to form a dimate and (iii) a chemical capable of completely hydrogenating the dimate to produce a fuel constituent; wherein the bioisoprene composition is produced by biological means.
 12. The system of claim 11, wherein the bioisoprene composition comprising greater than about 2 mg of isoprene and comprising greater than or about 99.94% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.
 13. The system of claim 11, wherein the one or more chemicals capable of dimerizing isoprene comprises catalyst for catalyzing cyclo-dimerization of isoprene comprising a catalyst selected from the group consisting of ruthenium catalysts, nickel catalysts, iron catalysts and chromium catalysts.
 14. The system of claim 11, wherein the catalyst for hydrogenating the unsaturated isoprene dimers comprises a catalyst selected from the group consisting of palladium catalysts, nickel catalysts, ruthenium catalysts and rhodium catalysts.
 15. The system of claim 11, wherein the chemical capable of partially hydrogenating isoprene comprises a palladium catalyst.
 16. The system of claim 11, wherein the chemical capable of dimerizing the isoamylene with mono-olefins comprises an acid catalyst.
 17. A fuel composition comprising a fuel constituent produced by the method of claim
 1. 18. The fuel composition of claim 17, wherein the fuel composition is substantially free of isoprene.
 19. The fuel composition of claim 17, wherein the fuel composition has δ¹³C value which is greater than −22‰ or within the range of −32‰ to −24‰.
 20. The method of claim 1, wherein at least about 80% of isoprene in the bioisoprene composition is converted to non-isoprene compounds.
 21. The method of claim 1, wherein the bioisoprene composition comprises one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.
 22. The method of claim 1, wherein the bioisoprene composition comprises one or more compounds selected from the group consisting of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, and C5 prenyl alcohols.
 23. The method of claim 1, wherein the bioisoprene composition comprises one or more compounds selected from the group consisting of 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine.
 24. The method of claim 1, wherein the isoprene in the bioisoprene composition is continuously chemically transformed to the non-isoprene compounds.
 25. The system of claim 11, wherein at least 80% of the isoprene in the bioisoprene composition is chemically converted to non-isoprene compounds.
 26. The system of claim 11, wherein the bioisoprene composition comprises one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.
 27. The system of claim 11, wherein the bioisoprene composition comprises one or more compounds selected from the group consisting of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, and C5 prenyl alcohols.
 28. The system of claim 11, wherein the bioisoprene composition comprises one or more compounds selected from the group consisting of 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. 